Therapeutic antiviral-wound healing compositions and methods for preparing and using same

ABSTRACT

This invention pertains to therapeutic wound healing compositions for protecting and resuscitating mammalian cells (Embodiment One (I)). This invention also pertains to therapeutic antiviral-wound healing compositions for reducing viral titers and increasing the proliferation and resuscitation rate of mammalian cells (Embodiment Two (II)). In a first aspect of Embodiment One (I.A), the therapeutic wound healing composition comprises (a) pyruvate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids. In a second aspect of Embodiment One (I.B), the therapeutic wound healing composition comprises (a) pyruvate, (b) lactate, and (c) a mixture of saturated and unsaturated fatty acids. In a third aspect of Embodiment One (I.C), the therapeutic wound healing composition comprises (a) an antioxidant and (b) a mixture of saturated and unsaturated fatty acids. In a fourth aspect of Embodiment One (I.D), the therapeutic wound healing composition comprises (a) lactate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids. In Embodiment Two (II), the therapeutic wound healing compositions of Embodiment One (I.A-D) are combined with a therapeutically effective amount of an antiviral agent (V) to form antiviral-wound healing compositions (II.A-D+V). This invention also pertains to methods for preparing and using the antiviral-wound healing compositions and the topical and ingestible pharmaceutical products in which the therapeutic compositions may be used.

REFERENCE TO RELATED UNITED STATES APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/224,936, filed Apr. 8, 1994, now abandoned, which is acontinuation-in-part of application Ser. No. 08/053,922, filed Apr. 26,1993, now abandoned which is a continuation of application Ser. No.07/663,500, filed Mar. 1, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to therapeutic wound healing compositions forprotecting and resuscitating mammalian cells (Embodiment One (I)). Thisinvention also pertains to therapeutic antiviral-wound healingcompositions for reducing viral titers and increasing the proliferationand resuscitation rate of mammalian cells (Embodiment Two (II)).

In Embodiment One (I), the therapeutic wound healing compositions areused alone. In a first aspect of Embodiment One (I.A), the therapeuticwound healing composition comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition comprises (a) anantioxidant and (b) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells. In a fourthaspect of Embodiment One (I.D), the therapeutic wound healingcomposition comprises (a) lactate selected from the group consisting oflactic acid, pharmaceutically acceptable salts of lactic acid, andmixtures thereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

In Embodiment Two (II), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a therapeutically effectiveamount of an antiviral agent (V) to form antiviral-wound healingcompositions (II.A-D+V). This invention also pertains to methods forpreparing and using the antiviral-wound healing compositions and thetopical and ingestible pharmaceutical products in which the therapeuticcompositions may be used.

2. Description of the Background

Wound Healing

Wounds are internal or external bodily injuries or lesions caused byphysical means, such as mechanical, chemical viral, bacterial, orthermal means, which disrupt the normal continuity of structures. Suchbodily injuries include contusions, wounds in which the skin isunbroken, incisions, wounds in which the skin is broken by a cuttinginstrument, and lacerations, wounds in which the skin is broken by adull or blunt instrument. Wounds may be caused by accidents or bysurgical procedures. Patients who suffer major wounds could benefit froman enhancement in the wound healing process.

Wound healing consists of a series of processes whereby injured tissueis repaired, specialized tissue is regenerated, and new tissue isreorganized.

Wound healing consists of three major phases: a) an inflammation phase(0-3 days), b) a cellular proliferation phase (3-12 days), and (c) aremodeling phase (3 days-6 months).

During the inflammation phase, platelet aggregation and clotting form amatrix which traps plasma proteins and blood cells to induce the influxof various types of cells. During the cellular proliferation phase, newconnective or granulation tissue and blood vessels are formed. Duringthe remodeling phase, granulation tissue is replaced by a network ofcollagen and elastin fibers leading to the formation of scar tissue.

When cells are injured or killed as a result of a wound, a wound healingstep is desirable to resuscitate the injured cells and produce new cellsto replace the dead cells. The healing process requires the reversal ofcytotoxicity, the suppression of inflammation, and the stimulation ofcellular viability and proliferation. Wounds require low levels ofoxygen in the initial stages of healing to suppress oxidative damage andhigher levels of oxygen in the later stages of healing to promotecollagen formation by fibroblasts.

Mammalian cells are continuously exposed to activated oxygen speciessuch as superoxide (O₂), hydrogen peroxide (H₂ O₂), hydroxyl radical(OH.), and singlet oxygen (¹ O₂). In vivo, these reactive oxygenintermediates are generated by cells in response to aerobic metabolism,catabolism of drugs and other xenobiotics, ultraviolet and x-rayradiation, and the respiratory burst of phagocytic cells (such as whiteblood cells) to kill invading bacteria such as those introduced throughwounds. Hydrogen peroxide, for example, is produced during respirationof most living organisms especially by stressed and injured cells.

These active oxygen species can injure cells. An important example ofsuch damage is lipid peroxidation which involves the oxidativedegradation of unsaturated lipids. Lipid peroxidation is highlydetrimental to membrane structure and function and can cause numerouscytopathological effects. Cells defend against lipid peroxidation byproducing radical scavengers such as superoxide dismutase, catalase, andperoxidase. Injured cells have a decreased ability to produce radicalscavengers. Excess hydrogen peroxide can react with DNA to causebackbone breakage, produce mutations, and alter and liberate bases.Hydrogen peroxide can also react with pyrimidines to open the 5,6-doublebond, which reaction inhibits the ability of pyrimidines to hydrogenbond to complementary bases, Hallaender et al. (1971). Such oxidativebiochemical injury can result in the loss of cellular membraneintegrity, reduced enzyme activity, changes in transport kinetics,changes in membrane lipid content, and leakage of potassium ions, aminoacids, and other cellular material.

Antioxidants have been shown to inhibit damage associated with activeoxygen species. For example, pyruvate and other α-ketoacids have beenreported to react rapidly and stoichiometrically with hydrogen peroxideto protect cells from cytolytic effects, O'Donnell-Tormey et al., J.Exp. Med., 165, pp. 500-514 (1987).

U.S. Pat. Nos. 3,920,835, 3,984,556, and 3,988,470, all issued to VanScott et al., disclose methods for treating acne, dandruff, and palmarkeratosis, respectively, which consist of applying to the affected areaa topical composition comprising from about 1% to about 20% of a loweraliphatic compound containing from two to six carbon atoms selected fromthe group consisting of α-hydroxyacids, α-ketoacids and esters thereof,and 3-hydroxybutryic acid in a pharmaceutically acceptable carrier. Thealiphatic compounds include pyruvic acid and lactic acid.

U.S. Pat. Nos. 4,105,783 and 4,197,316, both issued to Yu et al.,disclose a method and composition, respectively, for treating dry skinwhich consists of applying to the affected area a topical compositioncomprising from about 1% to about 20% of a compound selected from thegroup consisting of amides and ammonium salts of α-hydroxyacids,β-hydroxyacids, and α-ketoacids in a pharmaceutically acceptablecarrier. The compounds include the amides and ammonium salts of pyruvicacid and lactic acid.

U.S. Pat. No. 4,234,599, issued to Van Scott et al., discloses a methodfor treating actinic and nonactinic skin keratoses which consists ofapplying to the affected area a topical composition comprising aneffective amount of a compound selected from the group consisting ofα-hydroxyacids, β-hydroxyacids, and α-ketoacids in a pharmaceuticallyacceptable carrier. The acidic compounds include pyruvic acid and lacticacid.

U.S. Pat. No. 4,294,852, issued to Wildnauer et al., discloses acomposition for treating skin which comprises the α-hydroxyacids,β-hydroxyacids, and α-ketoacids disclosed above by Van Scott et al. incombination with C₃ -C₈ aliphatic alcohols.

U.S. Pat. No. 4,663,166, issued to Veech, discloses an electrolytesolution which comprises a mixture of L-lactate and pyruvate in a ratiofrom 20:1 to 1:1, respectively, or a mixture of D-β-hydroxybutyrate andacetoacetate, in a ratio from 6:1 to 0.5:1, respectively.

Sodium pyruvate has been reported to reduce the number of erosions,ulcers, and hemorrhages on the gastric mucosa in guinea pigs and ratscaused by acetylsalicylic acid. The analgesic and antipyretic propertiesof acetylsalicylic acid were not impaired by sodium pyruvate, Puschmann,Arzneimittelforschung, 33, pp. 410-415 and 415-416 (1983).

Pyruvate has been reported to exert a positive inotropic effect instunned myocardum, which is a prolonged ventricular dysfunctionfollowing brief periods of coronary artery occlusions which does notproduce irreversible damage, Mentzer et al., Ann. Surg., 209, pp.629-633 (1989).

Pyruvate has been reported to produce a relative stabilization of leftventricular pressure and work parameter and to reduce the size ofinfarctions. Pyruvate improves resumption of spontaneous beating of theheart and restoration of normal rates and pressure development, Bungeret al., J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki etal., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz et al.,Cardiovasc. Res., 15, pp.652-658 (1981), Giannelli et al., Ann. Thorac.Surg., 21, pp. 386-396 (1976).

Sodium pyruvate has been reported to act as an antagonist to cyanideintoxication (presumably through the formation of a cyanohydrin) and toprotect against the lethal effects of sodium sulfide and to retard theonset and development of functional, morphological, and biochemicalmeasures of acrylamide neuropathy of axons, Schwartz et al., Toxicol.Appl. Pharmacol., 50, pp. 437-442 (1979), Sabri etal., Brain Res., 483,pp. 1-11 (1989).

A chemotherapeutic cure of advanced L1210 leukemia has been reportedusing sodium pyruvate to restore abnormally deformed red blood cells tonormal. The deformed red blood cells prevented adequate drug delivery totumor cells, Cohen, Cancer Chemother. Pharmacol., 5, pp. 175-179 (1981).

Primary cultures of heterotopic tracheal transplant exposed in vivo to7,12-dimethyl-benz(a)anthracene were reported to be successfullymaintained in enrichment medium supplemented with sodium pyruvate alongwith cultures of interleukin-2 stimulated peripheral blood lymphocytes,and plasmacytomas and hybridomas, pig embryos, and human blastocysts,Shacter, J. Immunol. Methods, 99, pp. 259-270 (1987), Marchoketal.,Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil. Suppl.,33, pp. 115-124 (1985), Okamoto etal., No To Shinkei, 38, pp. 593-98(1986), Cohen et al., J. In Vitro Fert. Embryo Transfer, 2, pp. 59-64(1985).

U.S. Pat. Nos. 4,158,057, 4,351,835, 4,415,576, and 4,645,764, allissued to Stanko, disclose methods for preventing the accumulation offat in the liver of a mammal due to the ingestion of alcohol, forcontrolling weight in a mammal, for inhibiting body fat while increasingprotein concentration in a mammal, and for controlling the deposition ofbody fat in a living being, respectively. The methods compriseadministering to the mammal a therapeutic mixture of pyruvate anddihydroxyacetone, and optionally riboflavin. U.S. Pat. No. 4,548,937,issued to Stanko, discloses a method for controlling the weight gain ofa mammal which comprises administering to the mammal a therapeuticallyeffective amount of pyruvate, and optionally riboflavin. U.S. Pat. No.4,812,479, issued to Stanko, discloses a method for controlling theweight gain of a mammal which comprises administering to the mammal atherapeutically effective amount of dihydroxyacetone, and optionallyriboflavin and pyruvate.

Rats fed a calcium-oxalate lithogenic diet including sodium pyruvatewere reported to develop fewer urinary calculi (stones) than controlrats not given sodium pyruvate, Ogawa et al., Hinyokika Kiyo, 32, pp.1341-1347 (1986).

U.S. Pat. No. 4,521,375, issued to Houlsby, discloses a method forsterilizing surfaces which come into contact with living tissue. Themethod comprises sterilizing the surface with aqueous hydrogen peroxideand then neutralizing the surface with pyruvic acid.

U.S. Pat. No. 4,416,982, issued to Tauda et al., discloses a method fordecomposing hydrogen peroxide by reacting the hydrogen peroxide with aphenol or aniline derivative in the presence of peroxidase.

U.S. Pat. No. 4,696,917, issued to Lindstrom et al., discloses an eyeirrigation solution which comprises Eagle's Minimum Essential Mediumwith Earle's salts, chondroitin sulfate, a buffer solution,2-mercaptoethanol, and a pyruvate. The irrigation solution mayoptionally contain ascorbic acid and α-tocopherol. U.S. Pat. No.4,725,586, issued to Lindstrom et al., discloses an irrigation solutionwhich comprises a balanced salt solution, chondroitin sulfate, a buffersolution, 2-mercaptoethanol, sodium bicarbonate or dextrose, a pyruvate,a sodium phosphate buffer system, and cystine.

The irrigation solution may optionally contain ascorbic acid andgamma-tocopherol.

U.S. Pat. No. 3,887,702 issued to Baldwin, discloses a composition fortreating fingernails and toenails which consists essentially of soybeanoil or sunflower oil in combination with Vitamin E.

U.S. Pat. No. 4,847,069, issued to Bissett et al., discloses aphotoprotective composition comprising (a) a sorbohydroxamic acid, (b)an anti-inflammatory agent selected from steroidal anti-inflammatoryagents and a natural anti-inflammatory agent, and (c) a topical carrier.Fatty acids may be present as an emollient. U.S. Pat. No. 4,847,071,issued to Bissett et al., discloses a photoprotective compositioncomprising (a) a tocopherol or tocopherol ester radical scavenger, (b)an anti-inflammatory agent selected from steroidal anti-inflammatoryagents and a natural anti-inflammatory agent, and (c) a topical carrier.U.S. Pat. No. 4,847,072, issued to Bissett et al., discloses a topicalcomposition comprising not more than 25% tocopherol sorbate in a topicalcarrier.

U.S. Pat. No. 4,533,637, issued to Yamane et al., discloses a culturemedium which comprises a carbon source, a nucleic acid source precursor,amino acids, vitamins, minerals, a lipophilic nutrient, and serumalbumin, and cyclodextrins. The lipophilic substances includeunsaturated fatty acids and lipophilic vitamins such as Vitamin A, D,and E. Ascorbic acid may also be present.

United Kingdom patent application no. 2,196,348A, to Kovar et al.,discloses a synthetic culture medium which comprises inorganic salts,monosaccharides, amino acids, vitamins, buffering agents, and optionallysodium pyruvate adding magnesium hydroxide or magnesium oxide to theemulsion. The oil phase may include chicken fat.

U.S. Pat. No. 4,284,630, issued to Yu et al., discloses a method forstabilizing a water-in-oil emulsion which comprises adding magnesiumhydroxide or magnesium oxide to the emulsion. The oil phase may includechicken fat.

PREPARATION™ has been reported to increase the rate of wound healing inartificially created rectal ulcers. The active ingredients inPREPARATION H™ are skin respiratory factor and shark liver oil,Subramanyam et al., Digestive Diseases and Sciences, 29, pp. 829-832(1984).

The addition of sodium pyruvate to bacterial and yeast systems has beenreported to inhibit hydrogen peroxide production, enhance growth, andprotect the systems against the toxicity of reactive oxygenintermediates. The unsaturated fatty acids and saturated fatty acidscontained within chicken fat enhanced membrane repair and reducedcytotoxicity. The antioxidants glutathione and thioglycollate reducedthe injury induced by oxygen radical species, Martin, Ph.D. thesis,(1987-89).

U.S. Pat. No. 4,615,697, issued to Robinson, discloses a controlledrelease treatment composition comprising a treating agent and abioadhesive agent comprising a water-swellable but water-insoluble,fibrous crosslinked carboxy-functional polymer.

European patent application no. 0410696A1, to Kellaway et al., disclosesa mucoadhesive delivery system comprising a treating agent and apolyacrylic acid cross-linked with from about 1% to about 20% by weightof a polyhydroxy compound such as a sugar, cyclitol, or lower polyhydricalcohol.

Viral Infections

Herpes simplex virus type 1 (HSV-1) is a common viral infection inhumans which commonly causes epidermal lesions in and around the oralcavity. The hallmark of an HSV infection is the ability of the virus toestablish a latent infection in the nervous system, and to reactivateand cause recrudescent lesions. Recurrent disease can be a ratherunsightly, painful, and unpleasant episode, Overall J. C. Dermatologicviral diseases; In: Galasso GJ. Merigan TC. Buchanan RA. eds. Antiviralagents and viral diseases on man. 2nd ed. New York: Raven Press.1984:247-312.

The vast majority of perioral infections are caused by HSV type I andserologic studies indicate that 50% to 100% of the population hascontacted the virus by adulthood, Nahmias A. J., Roizman B., Infectionwith herpes-simplex virus 1 and 2, N. Engl. J. Med. 1973:289:781-789.More important, it is estimated that 20% to 45% of the population havereoccurring perioral HSV infections, most commonly in the form of feverblisters, Young S. K., 1 Rowe N H, Buchanan R. A., A clinical study forthe control of facial mucocutaneous herpes virus infections,I.Characterization of natural history in a professional schoolpopulation, Oral Surg. Oral Med. Oral Pathol., 1976:41:498-507; Embil J.A., Stephens R. G., Manuel F. R., Prevalence of recurrent herpeslabialis and aphthous ulcers among young adults on six continents, Can.Med.Assoc. J., 1975: 113:627-30; Ship I. I., Brightman V. J., Laster L.L., The patient with recurrent aphthous ulcers and the patient withrecurrent herpes labialis: a study of two population samples, J. Am.Dent. Assoc. 1967:75:645-54. Fever blisters are more than a minorannoyance; an estimated 98 million cases occur each year in the UnitedStates, Spruance S. L., Overall J. C. Jr, Kern E. R. et al., The naturalhistory of recurrent herpes simplex labialis: implications foranti-viral therapy, N. Eng. J. Med. 1977:197:69-75. Fever blisters causeconsiderable discomfort and are esthetically annoying to patients.

The Herpes group of viruses is composed of seven human viruses andmultiple animal viruses. The human herpes viruses consist of herpessimplex virus type I and II, varicella-zoster, cytomegalovirus,Epstein-barr, and human herpes virus types 6 and 7. The viruses aresimilar in size and morphology, and are characterized by adouble-stranded DNA core and a lipoprotein envelope with glycoproteinprojections. All the human herpes viruses replicate primarily in thecell nuclei. HSV-I and HSV-2 can be distinguished by a variety ofproperties, including clinical and epidemiologic patterns, antigenicity,DNA base composition, biologic characteristics, and sensitivity tovarious physical and chemical stresses, Rodu B., Russell C., MattinglyG., Determining therapeutic efficacy in recurrent herpes labialis bylesion size analysis, Oral Surg. Oral Med. Oral Pathol. Aug1991:178-182; Fox J. D., Briggs M., et al: Human herpes virus 6 insalivary glands, Lanced 336:590. 1990; Cory L., Spear P. G.: Infectionwith herpes simplex viruses (pts 1 and 2). N. Engl. J. of Med.314:686,749, 1986; Hammer S. M., et al.: Temporal cluster of herpessimplex encephalitis: investigation by restriction endonuclease cleavageof viral DNA, J. Infect. Dis. 141:436, 1980; Johnson R. E., Nahmias A.J., et al.: A seroepidemiologic survey of the prevalence of herpessimplex virus type 2 infection in the United States, N. Engl. J. Med.321:7, 1989.

HSV-I primary infections occur mainly in childhood. The herpes virus isa contact infectious agent that invades the moist membranes of the lips,mouth, or throat. The herpes virus is most frequently transmitted bykissing. Although virus titers are higher and transmission is morelikely when lesions are present, a symptomatic excretion of the virus iscommon. Thus, the virus may be transmitted even in the absence oflesions.

On entry into the skin sites, the virus replicates in epithelial cells,which results in lysis of infected cells and the instigation of a localinflammatory response. After primary infection, the virus may becomelatent within sensory nerve ganglion sites, Bonneau R. H., Sheridan J.F., et al. Stress-induction suppression of herpes simplex virus(HSV)-specific cytotoxic T lymphocyte and natural killer cell activityand enhancement of acute pathogenesis following local HSV infection,Brain, Behavior and Immunity 5, 170192, 1991; Rooney J. F., et al.Prevention of ultraviolet-light-induced herpes labialis by suncreen. TheLancet: 338:1419-1422.1991; Bastian F. O., Rabson A. S., Yee CL, et al.,Herpes virus hominis:isolation form human trigeminal ganglion, Science,1972: 178:306. In humans, the virus remains in a dormant state in thetrigeminal ganglion near the cheek bone. The virus can remain dormant inthese nerve cells for extended periods of time. The virus can emergefrom latency, track along the neural pathway back to the site of theoriginal infection causing the formation of a cold sore blister. Avariety of humoral and cell-mediated immune mechanisms are recruited inresponse to primary and recurrent HSV infections including theproduction of antibodies, interferon, activation of macrophages, theinduction of T-Lymphocyte-mediated reactivity, and the development ofantibody dependent lymphocyte cytotoxicity, Bonneau R. H., Sheridan JF,et al., Stress-induction suppression of herpes simplex virus(HSV)-specific cytotoxic T lymphocyte and natural killer cell activityand enhancement of acute pathogenesis following local HSV infection,Brain, Behavior and Immunity 5, 170192, 1991.

Once infected, the cold sore manifests itself in the form of a fluidfilled blister inside or outside the mouth. Other possible symptomswhich occur three to five days after exposure to the virus include:fever, swollen neck glands, and general aches and pain. As time passes,the fever blister will collapse followed by the formation of yellowcrust over the sore. The entire sore usually heals without scaringwithin two weeks, Bonneau R. H., Sheridan J. F., et al. Stress-inductionsuppression of herpes simplex virus (HSV)-specific cytotoxic Tlymphocyte and natural killer cell activity and enhancement of acutepathogenesis following local HSV infection, Brain, Behavior and Immunity5, 170192, 1991; Rooney J. F., et al. Prevention ofultraviolet-light-induced herpes labialis by suncreen. The Lancet:338:1419-1422.1991.

Recurrent infections are generally less severe than the primary attack.Recurrent infections decline in frequency after the age of 35. Signalsof recurrent infections include: itching, tingling, burning in the liparea, one to three days before the blister forms. In the United States,lip or perioral recurrences develop in 20 to 40% of the population.Recurrences vary in frequency from more than one attack per month toless than one attack every six months. Factors triggering recurrenceare: emotional stress, fever, illness, injury, overexposure to the sun,and menses. Sunlight triggers herpes labialis in approximately 25% ofpeople with recurrent infections.

The key to prevention is to eliminate exposure to the virus. When coldsores are present, one can prevent against autoinoculation by nottouching the sores. One can prevent against spreading the virus by notkissing other individuals. Currently cold sores cannot be cured.Treatment exists for relief of pain and discomfort. Antiseptic creams,emollients, and antiseptic ingredients reduce the discomfort throughtheir cooling and protective actions. Sun blocks with UVB protection actas a prophylaxis for people prone to recurrent cold sore sun blisters.

Several drugs are currently available for the treatment of HSVinfections. Acyclovir (trade name Zovirax) is a prescription compoundthat interferes with viral DNA replication through its action on viralthymidine kinase. Although extremely effective when given orally orintravenously for the treatment of primary or encephalitic HSVinfections, Acyclovir has little effectiveness, and is not generallyprescribed, for recurrent disease. A variety of over-the-countermedications are also available. Most of these medications rely on theweak antiviral properties of chemicals such as phenol which has a lowlevel of effectiveness against recurrent HSV infections, Whitley R. J.,Gnann J. W., Acyclovir a decade later, N. Eng. J. of Med. September1992,782-89.

While the above therapeutic wound healing compositions are reported toinhibit the production of reactive oxygen intermediates, none of theabove compositions are entirely satisfactory. None of the compositionshas the ability to simultaneously decrease cellular levels of hydrogenperoxide production, increase cellular resistance to cytotoxic agents,increase rates of cellular proliferation, increase cellular viability toprotect and resuscitate mammalian cells, and reduce virus titers. Thepresent invention provides such improved therapeutic antiviral-woundhealing compositions without the disadvantages characteristic ofpreviously known compositions. This invention also relates to methodsfor preparing and using the therapeutic antiviral-wound healingcompositions and the topical and ingestible pharmaceutical products inwhich the therapeutic compositions may be used.

SUMMARY OF THE INVENTION

This invention pertains to therapeutic wound healing compositions forprotecting and resuscitating mammalian cells (Embodiment One (I)). Thisinvention also pertains to therapeutic antiviral-wound healingcompositions for reducing viral titers and increasing the proliferationand resuscitation rate of mammalian cells (Embodiment Two (II)).

In Embodiment One (I), the therapeutic wound healing compositions areused alone. In a first aspect of Embodiment One (I.A), the therapeuticwound healing composition comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition comprises (a) anantioxidant and (b) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells. In a fourthaspect of Embodiment One (I.D), the therapeutic wound healingcomposition comprises (a) lactate selected from the group consisting oflactic acid, pharmaceutically acceptable salts of lactic acid, andmixtures thereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

Preferably, the wound healing composition comprises (I.A):

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

In Embodiment Two (II), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a therapeutically effectiveamount of an antiviral agent (V) to form antiviral-wound healingcompositions (II.A-D+V). The antiviral-wound healing compositions may beused alone or in combination with other medicaments. This invention alsopertains to methods for preparing and using the antiviral-wound healingcompositions and the topical and ingestible pharmaceutical products inwhich the therapeutic compositions may be used.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts in bar graph format the viability of U937 monocytic cellsfollowing exposure of the cells to various antioxidants (Examples 1-5).

FIG. 2 depicts in bar graph format the viability of U937 monocytic cellsfollowing exposure of the cells to various combinations of antioxidants(Examples 6-13).

FIG. 3 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious antioxidants (Examples 14-18).

FIG. 4 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious combinations of antioxidants (Examples 19-26).

FIG. 5 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids (Examples 27-32).

FIG. 6 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes following exposure of the cells tovarious antioxidants with and without a mixture of saturated andunsaturated fatty acids (Examples 33-42).

FIG. 7 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes following exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids (Examples 43-52).

FIG. 8 depicts in bar graph format a summary analysis of the levels ofhydrogen peroxide produced by epidermal keratinocytes following exposureof the cells to the individual components of the wound healingcomposition, to various combinations of the wound healing composition,and to the wound healing composition.

FIGS.9A-9D are photographs of wounded mice after 4 days of treatmentwith: no composition (FIG. 9A, control); a petrolatum base formulationcontaining live yeast cell derivative, shark oil, and a mixture ofsodium pyruvate, vitamin E, and chicken fat (FIG. 9B); a petrolatum baseformulation containing live yeast cell derivative and shark oil (FIG.9C); and Preparation H™ (FIG. 9D).

FIG. 10 is a photograph of a wounded mouse after 4 days of treatmentwith a petrolatum base formulation only (Example D).

FIG. 11 is a graph illustrating the lesion area curves for mice infectedwith herpes simplex virus and treated with acyclovir (ACV, positive) andpolyethylene glycol (PEG, negative) . The x-axis represents days postinfection and the y-axis represents the average lesion area (mm²).

FIG. 12 is a graph illustrating the symptom score curves for miceinfected with herpes simplex virus and treated with acyclovir (ACV,positive) and polyethylene glycol (PEG, negative) . The x-axisrepresents days post infection and the y-axis represents the symptomscore.

FIG. 13 is a graph illustrating the area under the symptom score curvesby group for mice infected with herpes simplex virus. The x-axisrepresents the groups and the y-axis represents the area under thesymptom score curve by day 12. The clinical symptoms for each group arerepresented as numbers on the x axis and the control groups(polyethylene glycol, base, or BLISTEX™) are represented by dottedlines.

FIGS. 14A-14B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 1.0 and 1.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 15A-15B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 2.0 and 2.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 16A-16B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 3.0 and 3.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 17A-17B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 4.0 and 0.0(control). The scorings range from 0 to 4, with 4 being the worst.

FIGS. 18A-18B are photographs illustrating the scoring of cold sorelesions in guinea pig. Animals in FIG. 48A were treated with formulas 11and 17. Animals in FIG. 48B were treated with Blistex™. The scoringsrange from 0 to 4, with 4 being the worst.

FIGS. 19A-19D are photographs illustrating the scoring of cold sorelesions in hairless mice. The scorings illustrated are 1.0, 2.0, 3.0 and4.0, respectively. The scorings range from 0 to 4, with 4 being theworst.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to therapeutic wound healing compositions forprotecting and resuscitating mammalian cells (Embodiment One (I)). Thisinvention also pertains to therapeutic antiviral-wound healingcompositions for reducing viral titers and increasing the proliferationand resuscitation rate of mammalian cells (Embodiment Two (II)).

In Embodiment One (I), the therapeutic wound healing compositions areused alone. In a first aspect of Embodiment One (I.A), the therapeuticwound healing composition comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition comprises (a) anantioxidant and (b) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells. In a fourthaspect of Embodiment One (I.D), the therapeutic wound healingcomposition comprises (a) lactate selected from the group consisting oflactic acid, pharmaceutically acceptable salts of lactic acid, andmixtures thereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

Preferably, the wound healing composition comprises (I.A):

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

In Embodiment Two (II), the therapeutic wound healing compositions ofEmbodiment One (I.A-D) are combined with a therapeutically effectiveamount of an antiviral agent (V) to form antiviral-wound healingcompositions (II.A-D+V). The antiviral-wound healing compositions may beused alone or in combination with other medicaments. This invention alsopertains to methods for preparing and using the antiviral-wound healingcompositions and the topical and ingestible pharmaceutical products inwhich the therapeutic compositions may be used.

As set out above, applicant has discovered therapeutic antiviral-woundhealing compositions (II.A-D+V) which comprise a therapeuticallyeffective amount of an antiviral agent (V) and the wound healingcompositions of Embodiment One (I.A-D). Antiviral agents can reducevirus titers in a patient but do not promote the wound healing process.Wound healing compositions can increase the resuscitation rate ofinjured mammalian cells and the proliferation rate of new mammaliancells to replace dead cells but do not reduce virus titers.

Applicant has found that the combination of an antiviral agent and awound healing composition results in a therapeutic antiviral-woundhealing composition which reduces the size, duration, and severity oforal and vaginal wounds suffered from viruses such as herpes. Cellstreated with the therapeutic wound healing compositions of the presentinvention show decreased levels of hydrogen peroxide production,increased resistance to cytotoxic agents, increased rates ofproliferation, and increased viability. Cellular cultures containing thetherapeutic wound healing compositions showed enhanced differentiationand proliferation over control cultures and rapidly formed attachmentsor tight junctions between the cells to form an epidermal sheet. Woundedmammals treated with the therapeutic antiviral-wound healingcompositions show significantly improved wound closing and healing overuntreated mammals and mammals treated with conventional healingcompositions.

The combination of the antiviral agent and the wound healingcompositions of the present invention provides a pharmaceuticalcomposition useful for reducing virus titers and having an enhancedability to prevent and reduce injury to mammalian cells and furtherincrease the resuscitation rate of injured mammalian cells. The tissuedamage associated with many viral diseases is believed to be caused bythe production of cellular produced active oxygen species. Combinationof the antiviral agent and the wound healing compositions may suppresssuch reactive oxygen-linked tissue injury.

The term "injured cell" as used herein means a cell that has anyactivity disrupted for any reason. For example, an injured cell may be acell that has injured membranes or damaged DNA, RNA, and ribosomes, forexample, a cell which has (a) injured membranes so that transportthrough the membranes is diminished resulting in an increase in toxinsand normal cellular wastes inside the cell and a decrease in nutrientsand other components necessary for cellular repair inside the cell, (b)an increase in concentration of oxygen radicals inside the cell becauseof the decreased ability of the cell to produce antioxidants andenzymes, or (c) damaged DNA, RNA, and ribosomes which must be repairedor replaced before normal cellular functions can be resumed. The term"resuscitation" of injured mammalian cells as used herein means thereversal of cytotoxicity, the stabilization of the cellular membrane, anincrease in the proliferation rate of the cell, and/or the normalizationof cellular functions such as the secretion of growth factors, hormones,and the like. The term "cytotoxicity" as used herein means a conditioncaused by a cytotoxic agent that injures the cell. Injured cells do notproliferate because injured cells expend all energy on cellular repair.Aiding cellular repair promotes cellular proliferation.

The term "prodrug", as used herein, refers to compounds which undergobiotransformation prior to exhibiting their pharmacological effects. Thechemical modification of drugs to overcome pharmaceutical problems hasalso been termed "drug latentiation." Drug latentiation is the chemicalmodification of a biologically active compound to form a new compoundwhich upon in vivo enzymatic attack will liberate the parent compound.The chemical alterations of the parent compound are such that the changein physicochemical properties will affect the absorption, distributionand enzymatic metabolism. The definition of drug latentiation has alsobeen extended to include nonenzymatic regeneration of the parentcompound. Regeneration takes place as a consequence of hydrolytic,dissociative, and other reactions not necessarily enzyme mediated. Theterms prodrugs, latentiated drugs, and bioreversible derivatives areused interchangeably. By inference, latentiation implies a time lagelement or time component involved in regenerating the bioactive parentmolecule in vivo. The term prodrug is general in that it includeslatentiated drug derivatives as well as those substances which areconverted after administration to the actual substance which combineswith receptors. The term prodrug is a generic term for agents whichundergo biotransformation prior to exhibiting their pharmacologicalactions. In the case where the administered drug is not the activeagent, but rather is biotransformed to the active agent, the term"prodrug" also includes compounds which may not necessarily undergobiotransformation to the administered drug but may undergobiotransformation to the active agent which exhibits the desiredpharmacological effect.

1. Wound Healing Compositions

A. Embodiment One (I.A-D)

The cells which may be treated with the therapeutic wound healingcompositions in the present invention are mammalian cells. Althoughapplicant will describe the present therapeutic wound healingcompositions as useful for treating mammalian epidermal keratinocytesand mammalian monocytes, applicant contemplates that the therapeuticwound healing compositions may be used to protect or resuscitate allmammalian cells. Keratinocytes are representative of normal mammaliancells and are the fastest proliferating cells in the body. Thecorrelation between the reaction of keratinocytes to injury and therapyand that of mammalian cells in general is very high. Monocytes arerepresentative of specialized mammalian cells such as the white bloodcells in the immune system and the organ cells in liver, kidney, heart,and brain. The mammalian cells may be treated in vivo and in vitro.

Epidermal keratinocytes are the specialized epithelial cells of theepidermis which synthesize keratin, a scleroprotein which is theprincipal constituent of epidermis, hair, nails, horny tissue, and theorganic matrix of the enamel of teeth. Mammalian epidermal keratinocytesconstitute about 95% of the epidermal cells and together withmelanocytes form the binary system of the epidermis. In its varioussuccessive stages, epidermal keratinocytes are also known as basalcells, prickle cells, and granular cells.

Monocytes are mononuclear phagocytic leukocytes which undergorespiratory bursting and are involved in reactive oxygen mediated damagewithin the epidermis. Leukocytes are white blood cells or corpuscleswhich may be classified into two main groups: granular leukocytes(granulocytes) which are leukocytes with abundant granules in thecytoplasm and nongranular leukocytes (nongranulocytes) which areleukocytes without specific granules in the cytoplasm and which includethe lymphocytes and monocytes. Phagocyte cells are cells which ingestmicroorganisms or other cells and foreign particles. Monocytes are alsoknown as large mononuclear leukocytes, and hyaline or transitionalleukocytes.

Epidermal keratinocytic cells and monocytic cells have multiple oxygengenerating mechanisms and the degree to which each type of mechanismfunctions differs in each type of cell. In monocytes, for example, therespiratory bursting process is more pronounced than in epidermalkeratinocytes. Hence, the components in the therapeutic wound healingcompositions of the present invention may vary depending upon the typesof cells involved in the condition being treated.

As set out above, in a first aspect of Embodiment One (I.A), thetherapeutic wound healing composition for treating mammalian cells,preferably epidermal keratinocytes, comprises (a) pyruvate selected fromthe group consisting of pyruvic acid, pharmaceutically acceptable saltsof pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids wherein the fatty acidsare those fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) an antioxidantand (b) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a fourth aspect ofEmbodiment One (I.D), the therapeutic wound healing composition fortreating mammalian cells, preferably monocytes, comprises (a) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells.

Pyruvic acid (2-oxopropanoic acid, α-ketopropionic acid, CH₃ COCOOH) orpyruvate is a fundamental intermediate in protein and carbohydratemetabolism and in the citric acid cycle. The citric acid cycle(tricarboxylic acid cycle, Kreb's cycle) is the major reaction sequencewhich executes the reduction of oxygen to generate adenosinetriphosphate (ATP) by oxidizing organic compounds in respiring tissuesto provide electrons to the transport system. Acetyl coenzyme A ("activeacetyl") is oxidized in this process and is thereafter utilized in avariety of biological processes and is a precursor in the biosynthesisof many fatty acids and sterols. The two major sources of acetylcoenzyme A are derived from the metabolism of glucose and fatty acids.Glycolysis consists of a series of transformations wherein each glucosemolecule is transformed in the cellular cytoplasm into two molecules ofpyruvic acid. Pyruvic acid may then enter the mitochondria where it isoxidized by coenzyme A in the presence of enzymes and cofactors toacetyl coenzyme A. Acetyl coenzyme A can then enter the citric acidcycle.

In muscle, pyruvic acid (derived from glycogen) can be reduced to lacticacid during anerobic metabolism which can occur during exercise. Lacticacid is reoxidized and partially retransformed to glycogen during rest.Pyruvate can also act as an antioxidant to neutralize oxygen radicals inthe cell and can be used in the multifunction oxidase system to reversecytotoxicity.

The pyruvate in the present invention may be selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, prodrugs of pyruvic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of pyruvic acid may be alkali saltsand alkaline earth salts. Preferably, the pyruvate is selected from thegroup consisting of pyruvic acid, lithium pyruvate, sodium pyruvate,potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate,manganese pyruvate, methyl pyruvate, α-ketoglutaric acid, and mixturesthereof. More preferably, the pyruvate is selected from the group ofsalts consisting of sodium pyruvate, potassium pyruvate, magnesiumpyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and thelike, and mixtures thereof. Most preferably, the pyruvate is sodiumpyruvate.

The amount of pyruvate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of pyruvate is that amount ofpyruvate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. The exact amount of pyruvate is a matter of preferencesubject to such factors as the type of condition being treated as wellas the other ingredients in the composition. In a preferred embodiment,pyruvate is present in the therapeutic wound healing composition in anamount from about 10% to about 50%, preferably from about 20% to about45%, and more preferably from about 25% to about 40%, by weight of thetherapeutic wound healing composition.

L-Lactic acid ((S)-2-hydroxypropanoic acid, (+) α-hydroxypropionic acid,CH₃ CHOHCOOH) or lactate occurs in small quantities in the blood andmuscle fluid of mammals. Lactic acid concentration increases in muscleand blood after vigorous activity. Lactate is a component in thecellular feedback mechanism and inhibits the natural respiratorybursting process of cells thereby suppressing the production of oxygenradicals.

The lactate in the present invention may be selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, prodrugs of lactic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of lactic acid may be alkali salts andalkaline earth salts. Preferably, the lactate is selected from the groupconsisting of lactic acid, lithium lactate, sodium lactate, potassiumlactate, magnesium lactate, calcium lactate, zinc lactate, manganeselactate, and the like, and mixtures thereof. More preferably, thelactate is selected from the group consisting of lactic acid, sodiumlactate, potassium lactate, magnesium lactate, calcium lactate, zinclactate, manganese lactate, and mixtures thereof. Most preferably, thelactate is lactic acid.

The amount of lactate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of lactate is that amount oflactate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. For an ingestible composition, a therapeuticallyeffective amount of lactate is that amount necessary to suppress therespiratory bursting process of white blood cells to protect andresuscitate the mammalian cells. In general, a therapeutically effectiveamount of lactate in an ingestible composition is from about 5 to about10 times the amount of lactate normally found in serum. The exact amountof lactate is a matter of preference subject to such factors as the typeof condition being treated as well as the other ingredients in thecomposition. In a preferred embodiment, lactate is present in thetherapeutic wound healing composition in an amount from about 10% toabout 50%, preferably from about 20% to about 45%, and more preferablyfrom about 25% to about 40%, by weight of the therapeutic wound healingcomposition.

Antioxidants are substances which inhibit oxidation or suppressreactions promoted by oxygen or peroxides. Antioxidants, especiallylipid-soluble antioxidants, can be absorbed into the cellular membraneto neutralize oxygen radicals and thereby protect the membrane. Theantioxidants useful in the present invention may be selected from thegroup consisting of all forms of Vitamin A including retinol and3,4-didehydroretinol, all forms of carotene such as α-carotene,β-carotene (beta, β-carotene), gamma-carotene, delta-carotene, all formsof Vitamin C (D-ascorbic acid, L-ascorbic acid), all forms of tocopherolsuch as Vitamin E (α-tocopherol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol),β-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone,tocotrienol, and Vitamin E esters which readily undergo hydrolysis toVitamin E such as Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate,prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and the like, and mixtures thereof. Preferably, theantioxidant is selected from the group of lipid-soluble antioxidantsconsisting of Vitamin A, β-carotene, Vitamin E, Vitamin E acetate, andmixtures thereof. More preferably, the antioxidant is Vitamin E orVitamin E acetate. Most preferably, the antioxidant is Vitamin Eacetate.

The amount of antioxidant present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of antioxidant is that amountof antioxidant necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of antioxidant is a matter ofpreference subject to such factors as the type of condition beingtreated as well as the other ingredients in the composition. In apreferred embodiment, the antioxidant is present in the therapeuticwound healing composition in an amount from about 0.1% to about 40%,preferably from about 0.2% to about 30%, and more preferably from about0.5% to about 20%, by weight of the therapeutic wound healingcomposition.

The mixture of saturated and unsaturated fatty acids in the presentinvention are those fatty acids required for the repair of mammaliancellular membranes and the production of new cells. Fatty acids arecarboxylic acid compounds found in animal and vegetable fat and oil.Fatty acids are classified as lipids and are composed of chains of alkylgroups containing from 4 to 22 carbon atoms and 0-3 double bonds andcharacterized by a terminal carboxyl group, --COOH. Fatty acids may besaturated or unsaturated and may be solid, semisolid, or liquid. Themost common saturated fatty acids are butyric acid (C₄), lauric acid(C₁₂), palmitic acid (C₁₆), and stearic acid (C₁₈). Unsaturated fattyacids are usually derived from vegetables and consist of alkyl chainscontaining from 16 to 22 carbon atoms and 0-3 double bonds with thecharacteristic terminal carboxyl group. The most common unsaturatedfatty acids are oleic acid, linoleic acid, and linolenic acid (all C₁₈acids).

In general, the mixture of saturated and unsaturated fatty acidsrequired for the repair of mammalian cellular membranes in the presentinvention may be derived from animal and vegetable fats and waxes,prodrugs of saturated and unsaturated fatty acids useful in the presentinvention, and mixtures thereof. For example, the fatty acids in thetherapeutic wound healing composition may be in the form of mono-, di-,or trigylcerides, or free fatty acids, or mixtures thereof, which arereadily available for the repair of injured cells. Cells produce thechemical components and the energy required for cellular viability andstore excess energy in the form of fat. Fat is adipose tissue storedbetween organs of the body to furnish a reserve supply of energy. Thepreferred animal fats and waxes have a fatty acid composition similar tothat of human fat and the fat contained in human breast milk. Thepreferred animal fats and waxes may be selected from the groupconsisting of human fat, chicken fat, cow fat (defined herein as abovine domestic animal regardless of sex or age), sheep fat, horse fat,pig fat, and whale fat. The more preferred animal fats and waxes may beselected from the group consisting of human fat and chicken fat. Themost preferred animal fat is human fat. Mixtures of other fats andwaxes, such as vegetable waxes (especially sunflower oil), marine oils(especially shark liver oil), and synthetic waxes and oils, which have afatty acid composition similar to that of animal fats and waxes, andpreferably to that of human fats and waxes, may also be employed.

In a preferred embodiment, the mixture of saturated and unsaturatedfatty acids has a composition similar to that of human fat and comprisesthe following fatty acids: butyric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, myristoleic acid, palmiticacid, palmitoleic acid, stearic, oleic acid, linoleic acid, linolenicacid, arachidic acid, and gaddoleic acid. Preferably, butyric acid,caproic acid, caprylic acid, capric acid, lauric acid, myristic acid,myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid,linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid arepresent in the mixture in about the following percentages by weight,respectively (carbon chain number and number of unsaturations are shownparenthetically, respectively): 0.2%-0.4% (C₄), 0.1% (C₆), 0.3%-0.8%(C₈), 2.2%-3.5% (C₁₀), 0.9%-5.5% (C₁₂), 2.8%-8.5% (C₁₄), 0.1%-0.6%(C_(14:1)), 23.2%-24.6% (C₁₆), 1.8%-3.0% (C_(16:1)), 6.9%-9.9% (C₁₈),36.0%-36.5% (C_(18:1)), 20%-20.6% (C_(18:2)), 7.5-7.8% (C_(18:3)),1.1%-4.9% (C₂₀), and 3.3%-6.4% (C_(20:1)).

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids is typically chicken fat comprising thefollowing fatty acids: lauric acid, myristic acid, myristoleic acid,pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid,margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid,arachidic acid, and gaddoleic acid. Preferably, lauric acid, myristicacid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleicacid, margaric acid, margaroleic acid, stearic, oleic acid, linoleicacid, linolenic acid, arachidic acid, and gaddoleic acid are present inthe mixture in about the following percentages by weight, respectively:0.1% (C₁₂), 0.8% (C₁₄), 0.2% (C_(14:1)), 0.1% (C₁₅), 25.3% (C₁₆), 7.2%(C_(16:1)), 0.1% (C₁₇), 0.1% (C_(17:1)), 6.5% (C₁₈), 37.7% (C_(18:1)),20.6% (C_(18:2)), 0.8% (C_(18:3)), 0.2% (C₂₀), and 0.3% (C_(20:1)), allpercentages ±10%.

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids comprises lecithin. Lecithin(phosphatidylcholine) is a phosphatide found in all living organisms(plants and animals) and is a significant constituent of nervous tissueand brain substance. Lecithin is a mixture of the diglycerides ofstearic, palmitic, and oleic acids, linked to the choline ester ofphosphoric acid. The product of commerce is predominantly soybeanlecithin obtained as a by-product in the manufacturing of soybean oil.Soybean lecithin contains palmitic acid 11.7%, stearic 4.0%, palmitoleic8.6%, oleic 9.8%, linoleic 55.0%, linolenic 4.0%, C₂₀ to C₂₂ acids(includes arachidonic) 5.5%. Lecithin may be represented by the formula:##STR1## wherein R is selected from the group consisting of stearic,palmitic, and oleic acid.

The above fatty acids and percentages thereof present in the fatty acidmixture are given as an example. The exact type of fatty acid present inthe fatty acid mixture and the exact amount of fatty acid employed inthe fatty acid 35 mixture may be varied in order to obtain the resultdesired in the final product and such variations are now within thecapabilities of those skilled in the art without the need for undueexperimentation.

The amount of fatty acids present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of fatty acids is that amountof fatty acids necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of fatty acids employed issubject to such factors as the type and distribution of fatty acidsemployed in the mixture, the type of condition being treated, and theother ingredients in the composition. In a preferred embodiment, thefatty acids are present in the therapeutic wound healing composition inan amount from about 10% to about 50%, preferably from about 20% toabout 45%, and more preferably from about 25% to about 40%, by weight ofthe therapeutic wound healing composition.

In accord with the present invention, the therapeutic wound healingcompositions of Embodiment One (I) for treating mammalian cells may beselected from the group consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(4) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Preferably, the wound healing compositions of Embodiment One (I) fortreating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes,comprise:

(I.A)

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably monocytes, comprise:

(I.D)

(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Throughout this disclosure, applicant will suggest various theories ormechanisms by which applicant believes the components in the therapeuticwound healing compositions and the antiviral agent function together inan unexpected synergistic manner to prevent and reduce injury tomammalian cells, increase the resuscitation rate of injured mammaliancells, and reduce viral titers. While applicant may offer variousmechanisms to explain the present invention, applicant does not wish tobe bound by theory. These theories are suggested to better understandthe present invention but are not intended to limit the effective scopeof the claims.

In the first aspect of Embodiment One (I.A), applicant believes thatpyruvate can be transported inside a cell where it can act as anantioxidant to neutralize oxygen radicals in the cell. Pyruvate can alsobe used inside the cell in the citric acid cycle to provide energy toincrease cellular viability, and as a precursor in the synthesis ofimportant biomolecules to promote cellular proliferation. In addition,pyruvate can be used in the multifunction oxidase system to reversecytotoxicity. Antioxidants, especially lipid-soluble antioxidants, canbe absorbed into the cell membrane to neutralize oxygen radicals andthereby protect the membrane. The saturated and unsaturated fatty acidsin the present invention are those fatty acids required for theresuscitation of mammalian cells and are readily available for therepair of injured cells and the proliferation of new cells. Cellsinjured by oxygen radicals need to produce unsaturated fatty acids torepair cellular membranes. However, the production of unsaturated fattyacids by cells requires oxygen. Thus, the injured cell needs high levelsof oxygen to produce unsaturated fatty acids and at the same time needsto reduce the level of oxygen within the cell to reduce oxidativeinjury. By providing the cell with the unsaturated fatty acids neededfor repair, the need of the cell for unsaturated fatty acids is reducedand the need for high oxygen levels is also reduced.

The combination of pyruvate inside the cell and an antioxidant in thecellular membrane functions in an unexpected synergistic manner toreduce hydrogen peroxide production in the cell to levels lower than canbe achieved by use of either type of component alone. The presence ofmixtures of saturated and unsaturated fatty acids in the therapeuticwound healing composition significantly enhances the ability of pyruvateand the antioxidant to inhibit reactive oxygen production. Bystabilizing the cellular membrane, unsaturated fatty acids also improvemembrane function and enhance pyruvate transport into the cell. Hence,the three components in the therapeutic wound healing composition of thefirst aspect of Embodiment One (I.A) function together in an unexpectedsynergistic manner to prevent and reduce injury to mammalian cells andincrease the resuscitation rate of injured mammalian cells.

In the second aspect of Embodiment One (I.B), lactate is employedinstead of an antioxidant. Antioxidants react with, and neutralize,oxygen radicals after the radicals are already formed. Lactate, on theother hand, is a component in the cellular feedback mechanism andinhibits the respiratory bursting process to suppress the production ofactive oxygen species. The combination of pyruvate to neutralize activeoxygen species and lactate to suppress the respiratory bursting processfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by use of either typeof component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing compositionsignificantly enhances the ability of pyruvate and lactate to inhibitreactive oxygen production. Hence, the three components in thetherapeutic wound healing composition in the second aspect of EmbodimentOne (I.B) function together in a synergistic manner to protect andresuscitate mammalian cells.

In the third aspect of Embodiment One (I.C), the presence of mixtures ofsaturated and unsaturated fatty acids in the therapeutic wound healingcomposition in this embodiment significantly enhances the ability of theantioxidant to inhibit reactive oxygen production. The combination of anantioxidant to neutralize active oxygen species and fatty acids torebuild cellular membranes and reduce the need of the cell for oxygenfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by either type ofcomponent alone. Hence, the components in the therapeutic wound healingcomposition in the third aspect of Embodiment One (I.C) functiontogether in a synergistic manner to protect and resuscitate mammaliancells.

In the fourth aspect of Embodiment One (I.D), lactate is employedbecause the respiratory bursting process is more pronounced in monocytesthan in epidermal keratinocytes. The combination of lactate to suppressthe respiratory bursting process and an antioxidant to neutralize activeoxygen species functions in a synergistic manner to reduce hydrogenperoxide production in the cell to levels lower than can be achieved byeither component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing composition inthis embodiment significantly enhances the ability of lactate and theantioxidant to inhibit reactive oxygen production. Hence, the threecomponents in the therapeutic wound healing composition in the fourthaspect of Embodiment One (I.D) function together in an unexpectedsynergistic manner to protect and resuscitate mammalian cells.

Accordingly, the combination of ingredients set out in the aboveembodiments functions together in an enhanced manner to prevent andreduce injury to mammalian cells and increase the resuscitation rate ofinjured mammalian cells. The therapeutic effect of the combination ofthe components in each of the above embodiments is markedly greater thanthat expected by the mere addition of the individual therapeuticcomponents. Hence, applicant's therapeutic wound healing compositionsfor treating mammalian cells have the ability to decrease intracellularlevels of hydrogen peroxide production, increase cellular resistance tocytotoxic agents, increase rates of cellular proliferation, and increasecellular viability.

B. Methods For Making The Wound Healing Compositions Of Embodiment One(I.A-D)

The present invention extends to methods for making the therapeuticwound healing compositions of Embodiment One (I.A-D). In general, atherapeutic wound healing composition is made by forming an admixture ofthe components of the composition. In a first aspect of Embodiment One(I.A), a therapeutic wound healing composition is made by forming anadmixture of (a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells. In a second aspect of Embodiment One (I.B), atherapeutic wound healing composition is made by forming an admixture of(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof,(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof,and (c) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a third aspect ofEmbodiment One (I.C), a therapeutic wound healing composition is made byforming an admixture of (a) an antioxidant and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a fourth aspect of Embodiment One(I.D), a therapeutic wound healing composition is made by forming anadmixture of (a) lactate selected from the group consisting of lacticacid, pharmaceutically acceptable salts of lactic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

For some applications, the admixture may be formed in a solvent such aswater, and a surfactant may be added if required. If necessary, the pHof the solvent is adjusted to a range from about 3.5 to about 8.0, andpreferably from about 4.5 to about 7.5, and more preferably about 6.0 toabout 7.4. The admixture is then sterile filtered. Other ingredients mayalso be incorporated into the therapeutic wound healing composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic wound healing composition (I.A) for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of admixingthe following ingredients:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells.

C. Methods For Employing The Wound Healing Compositions Of EmbodimentOne (I.A-D)

The present invention extends to methods for employing the therapeuticwound healing compositions of Embodiment One (I) in vivo and in vitro.In general, a therapeutic wound healing composition is employed bycontacting the therapeutic composition with mammalian cells.

In a first aspect of Embodiment One (I.A), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a second aspect of Embodiment One (I.B), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) lactate selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, and mixtures thereof, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the resuscitation of injured mammalian cells, and (B)contacting the therapeutic wound healing composition with the mammaliancells.

In a third aspect of Embodiment One (I.C), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a fourth aspect of Embodiment One (I.D), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) lactate selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a preferred embodiment, the invention is directed to a method forhealing a wound in a mammal which comprises the steps of:

(A) providing a therapeutic wound healing composition (I.A) whichcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells; and

(B) contacting the therapeutic wound healing composition with the wound.

The types of wounds which may be healed using the wound healingcompositions of Embodiment One (I.A-D) of the present invention arethose which result from an injury which causes epidermal damage such asincisions, wounds in which the skin is broken by a cutting instrument,and lacerations, wounds in which the skin is broken by a dull or bluntinstrument. The therapeutic compositions may also be used to treatvarious dermatological disorders such as hyperkeratosis, photo-aging,burns, donor site wounds from skin transplants, ulcers (cutaneous,decubitis, venous stasis, and diabetic), psoriasis, skin rashes, andsunburn photoreactive processes. The topical therapeutic compositionsmay also be used orally in the form of a mouth wash or spray to protectand accelerate the healing of injured oral tissue such as mouth soresand burns. The topical therapeutic compositions may further be used inophthalmological preparations to treat wounds such as those which resultfrom corneal ulcers, radialkeratotomy, corneal transplants,epikeratophakia and other surgically induced wounds in the eye. Thetopical therapeutic compositions may in addition be used in anorectalcreams and suppositories to treat such conditions as pruritus ani,proctitis, anal fissures, and hemorrhoids. In a preferred embodiment,the therapeutic compositions are used to treat wounds such as incisionsand lacerations.

The wound healing compositions of Embodiment One (I.A-D) of the presentinvention may be utilized in topical products, ingestible products, andtissue culture medium to protect mammalian cells and increase theresuscitation rate of injured mammalian cells. For example, thetherapeutic wound healing compositions may be used in topical skin careproducts to protect and increase the resuscitation rate of skin tissuesuch as in the treatment of various dermatological disorders such ashyperkeratosis, photo-aging, and sunburn photoreactive processes. Injuryto skin can occur for a variety of reasons. Injury often occurs toindividuals who wash their hands often, to individuals who are exposedto stressful environmental conditions (overexposure to sun orchemicals), or to the elderly or individuals with an underliningdisease. The addition of the wound healing compositions of the presentinvention to a lotion provides a source of antioxidants to the skinwhich would protect the skin from the harmful effects of UV light,chemicals, and severe drying. The wound healing compositions can be usedfor the following indications: a) Moisturizing and protecting; b)Healing dry cracked skin; c) Treating irritated skin such as diaperrash; d) Healing severe dry skin due to other diseases (venousdermatitis); e) Treating psoriasis and other hyperproliferativediseases; f) Protecting skin from UV light damage (antioxidant skinreplacement); g) Treating seborrheic conditions; and h) Treating shavingwounds in an after shave lotion.

The topical therapeutic wound healing compositions may also be usedorally in the form of a mouth wash or spray to protect and acceleratethe healing of injured oral tissue such as mouth sores and burns. Thetopical therapeutic wound healing compositions may further be used inophthalmological preparations such as eye care products to neutralizehydrogen peroxide used in the cleaning of contact lenses. The topicaltherapeutic wound healing compositions may in addition be used inanorectal creams and suppositories to treat such conditions as pruritusani, proctitis, anal fissures, and hemorrhoids. Initially as white bloodcells enter a wound site, the cells release oxygen radicals, depletingthe antioxidants at the wound site, thus impairing the healing process.Incorporating the wound healing compositions of the present inventioninto a wound healing formulation would facilitate healing by providingthe site with usable antioxidants, and a source of fatty acids neededfor membrane repair. The wound healing compositions can be used for thefollowing indications: a) Healing of cuts and scrapes; b) Burns (healsburns with less scaring and scabbing); c) Decubitus ulcers; d) Bedsores, pressure ulcers; e) Fissures, Hemorrhoids; f) Use in combinationwith immunostimulators (simulated healing in healing deficient people);g) Post surgical wounds; h) Bandages; i) Diabetic ulcers; j) Venousulceration; and k) Use in combination with wound cleansing agents.

The therapeutic wound healing compositions may also be used iningestible products to protect and increase the resuscitation rate oferosions, stomach ulcers, and hemorrhages in the gastric mucosa. Otheringestible therapeutic products include: stroke medications; autoimmunedisease medications; arthritis medications; ulcer medications; cancermedications (cytotoxic agents);

heart medication to improve regional ventricular function and restorenormal heart rate and pressure functions; lung medication to repairinjured tissue; liver medication to suppress lipogenesis of alcoholicorigin and prevent hepatic steatosis; kidney medication to suppressurinary calculi (kidney stones); detoxification medication to antagonizeheavy metal poisoning, cyanide poisoning, sodium sulfide poisoning,other types of poisoning, and reduce and neutralize the production ofoxygen radicals which produces injury to tissue, to protect and furtherenhance the resuscitation rate of the injured mammalian cells. Thetherapeutic wound healing compositions may be used in ingestibleproducts to treat inflammatory diseases such as hepatitis, gastritis,colitis, esophagitis, arthritis, and pancreatitis.

The therapeutic wound healing compositions of the present invention mayalso be used in tissue culture media and organ transplant media toprevent and reduce injury to mammalian cells and increase theresuscitation rate of injured mammalian cells. Tissue cultures andtransplant organs encounter reactive oxygen species generated in theculture media by the injured cells. Organs particularly susceptible tooxidative damage during transport and transplantation due to reperfusioninjury following ischemia are corneas, livers, hearts, and kidneys. Thetherapeutic wound healing compositions may be useful to abrogatereperfusion injury to such transplant organs.

In a specific embodiment, the invention is directed to a method forpreserving mammalian cells in a culture medium which comprises the stepsof:

(A) providing a therapeutic wound healing composition selected from thegroup of consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(4) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells;

(B) providing mammalian cells in a culture medium; and

(C) contacting the therapeutic wound healing composition from step (A)with the mammalian cells in the culture medium from step (B).

D. Formulations Of The Wound Healing Compositions Of Embodiment One(I.A-D)

Once prepared, the inventive therapeutic wound healing compositions ofEmbodiment One (I.A-D) may be stored for future use or may be formulatedin effective amounts with pharmaceutically acceptable carriers toprepare a wide variety of pharmaceutical compositions. Examples ofpharmaceutically acceptable carriers are pharmaceutical appliances,topical vehicles (non-oral and oral), and ingestible vehicles.

Examples of pharmaceutical appliances are sutures, staples, gauze,bandages, bum dressings, artificial skins, liposome or micellformulations, microcapsules, aqueous vehicles for soaking gauzedressings, and the like, and mixtures thereof. Non-oral topicalcompositions employ non-oral topical vehicles, such as creams, gelsformulations, foams, ointments and sprays, salves, and films, which areintended to be applied to the skin or body cavity and are not intendedto be taken by mouth. Oral topical compositions employ oral vehicles,such as mouthwashes, rinses, oral sprays, suspensions, and dental gels,which are intended to be taken by mouth but are not intended to beingested. Ingestible compositions employ ingestible or partly ingestiblevehicles such as confectionery bulking agents which include hard andsoft confectionery such as lozenges, tablets, toffees, nougats,suspensions, chewy candies, and chewing gums.

In one form of the invention, the therapeutic wound healing compositionis incorporated into a pharmaceutical appliance which may be in the formof sutures, staples, gauze, bandages, burn dressings, artificial skins,liposome or micell formulations, microcapsules, aqueous vehicles forsoaking gauze dressings, and the like, and mixtures thereof. A varietyof traditional ingredients may optionally be included in thepharmaceutical composition in effective amounts such as buffers,preservatives, tonicity adjusting agents, antioxidants, polymers foradjusting viscosity or for use as extenders, and excipients, and thelike. Specific illustrative examples of such traditional ingredientsinclude acetate and borate buffers; thimerosol, sorbic acid, methyl andpropyl paraben and chlorobutanol preservatives; sodium chloride andsugars to adjust the tonicity; and excipients such as mannitol, lactoseand sucrose. Other conventional pharmaceutical additives known to thosehaving ordinary skill in the pharmaceutical arts may also be used in thepharmaceutical composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe employed in the pharmaceutical appliance. These amounts are readilydetermined by those skilled in the art without the need for undueexperimentation. The exact amount of the therapeutic wound healingcomposition employed is subject to such factors as the type andconcentration of the therapeutic wound healing composition and the typeof pharmaceutical appliance employed. Thus, the amount of therapeuticwound healing composition may be varied in order to obtain the resultdesired in the final product and such variations are within thecapabilities of those skilled in the art without the need for undueexperimentation. In a preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, by weight of the pharmaceuticalcomposition. In a more preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 3%, by weight of the pharmaceuticalcomposition. In a most preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 1%, by weight of the pharmaceuticalcomposition.

The present invention extends to methods for making the pharmaceuticalcompositions. In general, a pharmaceutical composition is made bycontacting a therapeutically effective amount of a therapeutic woundhealing composition with a pharmaceutical appliance and the otheringredients of the final desired pharmaceutical composition. Thetherapeutic wound healing composition may be in a solvent and may beabsorbed onto a pharmaceutical appliance.

Other ingredients will usually be incorporated into the composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate pharmaceuticalcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into a non-oral topical vehicle which may bein the form of a cream, gel, foam, ointment, spray, and the like.Typical non-toxic non-oral topical vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred non-oraltopical vehicles are water and pharmaceutically acceptablewater-miscible organic solvents such as ethyl alcohol, isopropylalcohol, propylene glycol, glycerin, and the like, and mixtures of thesesolvents. Water-alcohol mixtures are particularly preferred and aregenerally employed in a weight ratio from about 1:1 to about 20:1,preferably from about 3:1 to about 20:1, and most preferably from about3:1 to about 10:1, respectively.

The non-oral topical therapeutic wound healing compositions may alsocontain conventional additives employed in those products. Conventionaladditives include humectants, emollients, lubricants, stabilizers, dyes,and perfumes, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

Suitable humectants useful in the non-oral topical therapeutic woundhealing compositions include glycerin, propylene glycol, polyethyleneglycol, sorbitan, fructose, and the like, and mixtures thereof.Humectants, when employed, may be present in amounts from about 10% toabout 20%, by weight of the topical therapeutic wound healingcomposition.

The coloring agents (colors, colorants) useful in the non-oral topicaltherapeutic wound healing composition are used in amounts effective toproduce the desired color. These coloring agents include pigments whichmay be incorporated in amounts up to about 6% by weight of the non-oraltopical therapeutic wound healing composition. A preferred pigment,titanium dioxide, may be incorporated in amounts up to about 2%, andpreferably less than about 1%, by weight of the non-oral topicaltherapeutic wound healing composition. The coloring agents may alsoinclude natural food colors and dyes suitable for food, drug andcosmetic applications. These coloring agents are known as F.D.& C. dyesand lakes. The materials acceptable for the foregoing uses arepreferably water-soluble. Illustrative nonlimiting examples include theindigoid dye known as F.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo.1 comprises a triphenylmethane dye and is the monosodium salt of 4-4-(N-ethyl-p-sulfoniumbenzylamino) diphenylmethylene!-1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine!. A fullrecitation of all F.D.& C. coloring agents and their correspondingchemical structures may be found in the Kirk-Othmer Encyclopedia ofChemical Technology, 3rd Edition, in volume 5 at pages 857-884, whichtext is incorporated herein by reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with a non-oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.In a more preferred embodiment, the non-oral topical therapeutic woundhealing compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 2%, and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the non-oraltopical therapeutic wound healing compositions. In such a method, thenon-oral topical therapeutic wound healing composition is prepared byadmixing a therapeutically effective amount of the therapeutic woundhealing composition of the present invention and a non-oral topicalvehicle. The final compositions are readily prepared using standardmethods and apparatus generally known by those skilled in thepharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into an oral topical vehicle which may be inthe form of a mouthwash, rinse, oral spray, suspension, dental gel, andthe like. Typical non-toxic oral vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred oral vehiclesare water, ethanol, and water-ethanol mixtures. The water-ethanolmixtures are generally employed in a weight ratio from about 1:1 toabout 20:1, preferably from about 3:1 to about 20:1, and most preferablyfrom about 3:1 to about 10:1, respectively. The pH value of the oralvehicle is generally from about 4 to about 7, and preferably from about5 to about 6.5. An oral topical vehicle having a pH value below about 4is generally irritating to the oral cavity and an oral vehicle having apH value greater than about 7 generally results in an unpleasant mouthfeel.

The oral topical therapeutic wound healing compositions may also containconventional additives normally employed in those products. Conventionaladditives include a fluorine providing compound, a sweetening agent, aflavoring agent, a coloring agent, a humectant, a buffer, and anemulsifier, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

The coloring agents and humectants, and the amounts of these additivesto be employed, set out above as useful in the non-oral topicaltherapeutic wound healing composition may be used in the oral topicaltherapeutic wound healing composition.

Fluorine providing compounds may be fully or slightly water soluble andare characterized by their ability to release fluoride ions or fluoridecontaining ions in water and by their lack of reaction with othercomponents in the composition. Typical fluorine providing compounds areinorganic fluoride salts such as water-soluble alkali metal, alkalineearth metal, and heavy metal salts, for example, sodium fluoride,potassium fluoride, ammonium fluoride, cuprous fluoride, zinc fluoride,stannic fluoride, stannous fluoride, barium fluoride, sodiumfluorosilicate, ammonium fluorosilicate, sodium fluorozirconate, sodiummonofluorophosphate, aluminum mono- and di-fluorophosphates andfluorinated sodium calcium pyrophosphate. Alkali metal fluorides, tinfluoride and monofluorophosphates, such as sodium and stannous fluoride,sodium monofluorophosphate and mixtures thereof, are preferred.

The amount of fluorine providing compound present in the present oraltopical therapeutic wound healing composition is dependent upon the typeof fluorine providing compound employed, the solubility of the fluorinecompound, and the nature of the final oral therapeutic wound healingcomposition. The amount of fluorine providing compound used must be anontoxic amount. In general, the fluorine providing compound when usedwill be present in an amount up to about 1%, preferably from about0.001% to about 0.1%, and most preferably from about 0.001% to about0.05%, by weight of the oral topical therapeutic wound healingcomposition.

When sweetening agents (sweeteners) are used, those sweeteners wellknown in the art, including both natural and artificial sweeteners, maybe employed. The sweetening agent used may be selected from a wide rangeof materials including water-soluble sweetening agents, water-solubleartificial sweetening agents, water-soluble sweetening agents derivedfrom naturally occurring water-soluble sweetening agents, dipeptidebased sweetening agents, and protein based sweetening agents, includingmixtures thereof. Without being limited to particular sweetening agents,representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribose, glucose(dextrose), mannose, galactose, fructose (levulose), sucrose (sugar),maltose, invert sugar (a mixture of fructose and glucose derived fromsucrose), partially hydrolyzed starch, corn syrup solids,dihydrochalcones, monellin, steviosides, and glycyrrhizin, and mixturesthereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspartame)and materials described in U.S. Pat. No. 3,492,131,L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amide hydrate(Alitame), methyl esters of L-aspartyl-L-phenylglycerine andL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), e.g., chlorodeoxysugar derivatives such as derivativesof chlorodeoxysucrose or chlorodeoxygalactosucrose, known, for example,under the product designation of Sucralose; examples ofchlorodeoxysucrose and chlorodeoxygalacto-sucrose derivatives includebut are not limited to: 1-chloro-1'-deoxysucrose;4-chloro-4deoxy-α-D-galacto-pyranosyl-α-D-fructofuranoside, or4-chloro-4-deoxygalactosucrose;4-chloro-4-deoxy-α-D-galacto-pyranosyl-1-chloro-1-deoxy-β-D-fructo-furanoside,or 4,1'-dichloro-4,1'-dideoxygalactosucrose;1',6'-dichloro-1',6'-dideoxysucrose;4-chloro-4-deoxy-α-D-galacto-pyranosyl-1,6-dichloro-1,6-dideoxy-.beta.-D-fructo-furanoside,or 4,1',6'-trichloro4,1',6'-trideoxygalacto-sucrose;4,6-dichloro-4,6-dideoxy-α-D-galacto-pyranosyl-6-chloro-6-deoxy-β-D-fructofuranoside,or 4,6,6'-trichloro-4,6,6'-trideoxygalactosucrose;6,1',6'-trichloro-6,1',6'-trideoxysucrose;4,6-dichloro-4,6-dideoxy-α-D-galacto-pyranosyl-1,6-dichloro-1,6-di-deoxy-β-D-fructofuranoside,or 4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxygalacto-sucrose; and4,6,1',6'-tetrachloro4,6, 1',6'-tetradeoxy-sucrose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand II).

In general, an effective amount of sweetening agent is utilized toprovide the level of sweetness desired in the particular oral topicaltherapeutic wound healing composition, and this amount will vary withthe sweetener selected and the final oral therapeutic product desired.The amount of sweetener normally present is in the range from about0.0025% to about 90%, by weight of the oral topical therapeutic woundhealing composition, depending upon the sweetener used.

The exact range of amounts for each type of sweetener is well known inthe art and is not the subject of the present invention.

The flavoring agents (flavors, flavorants) which may be used includethose flavors known to the skilled artisan, such as natural andartificial flavors. Suitable flavoring agents include mints, such aspeppermint, citrus flavors such as orange and lemon, artificial vanilla,cinnamon, various fruit flavors, both individual and mixed, and thelike.

The amount of flavoring agent employed in the oral topical therapeuticwound healing composition is normally a matter of preference subject tosuch factors as the type of final oral therapeutic wound healingcomposition, the individual flavor employed, and the strength of flavordesired. Thus, the amount of flavoring may be varied in order to obtainthe result desired in the final product and such variations are withinthe capabilities of those skilled in the art without the need for undueexperimentation. The flavoring agents, when used, are generally utilizedin amounts that may, for example, range in amounts from about 0.05% toabout 6%, by weight of the oral topical therapeutic wound healingcomposition.

Suitable buffer solutions useful in the non-oral topical therapeuticwound healing compositions include citric acid-sodium citrate solution,phosphoric acid-sodium phosphate solution, and acetic acid-sodiumacetate solution in amounts up to about 1%, and preferably from about0.05% to about 0.5% by weight of the oral topical therapeutic woundhealing composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with an oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a oral topical vehicle in aquantity sufficient to bring the total amount of composition to 100%, byweight of the oral topical therapeutic wound healing composition. In amore preferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the oral topical therapeutic wound healing compositions willcomprise the therapeutic wound healing composition in an amount fromabout 0.1% to about 2%, and a oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the oral topicaltherapeutic wound healing compositions. In such a method, the oraltopical therapeutic wound healing composition is prepared by admixing atherapeutically effective amount of the therapeutic wound healingcomposition of the present invention and an oral topical vehicle. Thefinal compositions are readily prepared using standard methods andapparatus generally known by those skilled in the pharmaceutical arts.The apparatus useful in accordance with the present invention comprisesmixing apparatus well known in the pharmaceutical arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.

In a preferred embodiment, an oral topical therapeutic wound healingcomposition is made by first dissolving coloring agents, sweeteningagents, and similar additives in water. The therapeutic wound healingcomposition is then admixed with the aqueous solution. Then sufficientwater or ethanol, or mixtures of water and ethanol, are added to thesolution with mixing until the final solution volume is reached. In amore preferred embodiment, the therapeutic wound healing composition isadded to the solution as the final ingredient. The final oral topicaltherapeutic wound healing compositions are readily prepared usingmethods generally known in the pharmaceutical arts.

The oral therapeutic wound healing composition may also be in the formof dental gel. As used herein, the term "gel" means a solid or semisolidcolloid which contains considerable quantities of water. The colloidparticles in a gel are linked together in a coherent meshwork whichimmobilizes the water contained inside the meshwork.

The dental gel compositions of the present invention may contain theconventional additives set out above for oral topical therapeutic woundhealing compositions such as mouthwashes, rinses, oral sprays, andsuspensions and, in addition, may contain additional additives such as apolishing agent, a desensitizing agent, and the like, providing theadditional additives do not interfere with the therapeutic properties ofthe therapeutic wound healing composition.

In a dental gel composition, the oral vehicle generally comprises water,typically in an amount from about 10% to about 90%, by weight of thedental gel composition. Polyethylene glycol, propylene glycol, glycerin,and mixtures thereof may also be present in the vehicle as humectants orbinders in amounts from about 18% to about 30%, by weight of the dentalgel composition. Particularly preferred oral vehicles comprise mixturesof water with polyethylene glycol or water with glycerin andpolypropylene glycol.

The dental gels of the present invention include a gelling agent(thickening agent) such as a natural or synthetic gum or gelatin.Gelling agents such as hydroxyethyl cellulose, methyl cellulose,glycerin, carboxypolymethylene, and gelatin and the like, and mixturesthereof may be used. The preferred gelling agent is hydroxyethylcellulose. Gelling agents may be used in amounts from about 0.5% toabout 5%, and preferably from about 0.5% to about 2%, by weight of thedental gel composition.

The dental gel compositions of the present invention may also include apolishing agent. In clear gels, a polishing agent of colloidal silicaand/or alkali metal aluminosilicate complexes is preferred since thesematerials have refractive indices close to the refractive indices of thegelling systems commonly used in dental gels. In non-clear gels, apolishing agent of calcium carbonate or calcium dihydrate may be used.These polishing agents may be used in amounts up to about 75%, andpreferably in amounts up to about 50%, by weight of the dental gelcomposition.

The dental gel may also contain a desensitizing agent such as acombination of citric acid and sodium citrate. Citric acid may be usedin an amount from about 0.1% to about 3%, and preferably from about 0.2%to about 1%, by weight, and sodium citrate may be used in an amount fromabout 0.3% to about 9%, and preferably from about 0.6% to about 3%, byweight of the dental gel composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the dental gel compositions. These amounts are readilydetermined by those skilled in the art without the need for undueexperimentation. In a preferred embodiment, the dental gel compositionswill comprise the therapeutic wound healing composition in an amountfrom about 0.1% to about 10% and an oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the dental gel composition. In a more preferred embodiment, thedental gel compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the dental gel compositions will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and an oral topical vehicle in a quantity sufficient to bringthe total amount of composition to 100%, by weight of the dental gelcomposition.

The present invention extends to methods for preparing the therapeuticdental gel compositions. In such a method, the dental gel composition isprepared by admixing a therapeutically effective amount of thetherapeutic wound healing composition of the present invention and anoral topical vehicle. The final compositions are readily prepared usingmethods generally known by those skilled in the dental andpharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In a preferred embodiment, a therapeutic dental gel composition is madeby first dispersing a gelling agent in a humectant or water, or amixture of both, then admixing to the dispersion an aqueous solution ofthe water-soluble additives such as the fluorine providing compound,sweeteners and the like, then adding the polishing agent, and lastlyadmixing the flavoring agent and the therapeutic wound healingcomposition. The final gel mixture is then tubed or otherwise packaged.The liquids and solids in a gel product are proportioned to form acreamy or gelled mass which is extrudable from a pressurized containeror from a collapsible tube. The final therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In yet another form of the invention, the therapeutic wound healingcomposition is incorporated into an ingestible vehicle. The ingestiblevehicle may be a confectionery bulking agent in the form of lozenges,tablets, toffees, nougats, suspensions, chewy candies, chewing gums, andthe like. The pharmaceutically acceptable carriers may be prepared froma wide range of materials including, but not limited to, diluents,binders and adhesives, lubricants, disintegrants, coloring agents,bulking agents, flavoring agents, sweetening agents and miscellaneousmaterials such as buffers and adsorbents that may be needed in order toprepare a particular therapeutic confection.

The preparation of confectionery formulations is historically well knownand has changed little through the years. Confectionery items have beenclassified as either "hard" confectionery or "soft" confectionery. Thetherapeutic wound healing compositions of the present invention can beincorporated into confectionery compositions by admixing the inventivecomposition into conventional hard and soft confections.

As used herein, the term confectionery material means a productcontaining a bulking agent selected from a wide variety of materialssuch as sugar, corn syrup, and in the case of sugarless bulking agents,sugar alcohols such as sorbitol and mannitol and mixtures thereof.Confectionery material may include such exemplary substances aslozenges, tablets, toffee, nougat, suspensions, chewy candy, chewing gumand the like. The bulking agent is present in a quantity sufficient tobring the total amount of composition to 100%. In general, the bulkingagent will be present in amounts up to about 99.98%, preferably inamounts up to about 99.9%, and more preferably in amounts up to about99%, by weight of the ingestible therapeutic wound healing composition.

Lozenges are flavored medicated dosage forms intended to be sucked andheld in the mouth. Lozenges may be in the form of various shapes such asflat, circular, octagonal and biconvex forms. The lozenge bases aregenerally in two forms: hard boiled candy lozenges and compressed tabletlozenges.

Hard boiled candy lozenges may be processed and formulated byconventional means. In general, a hard boiled candy lozenge has a basecomposed of a mixture of sugar and other carbohydrate bulking agentskept in an amorphous or glassy condition. This amorphous or glassy formis considered a solid syrup of sugars generally having from about 0.5%to about 1.5% moisture. Such materials normally contain up to about 92%corn syrup, up to about 55% sugar and from about 0.1% to about 5% water,by weight of the final composition. The syrup component is generallyprepared from corn syrups high in fructose, but may include othermaterials. Further ingredients such as flavoring agents, sweeteningagents, acidulants, coloring agents and the like may also be added.

Boiled candy lozenges may also be prepared from non-fermentable sugarssuch as sorbitol, mannitol, and hydrogenated corn syrup. Typicalhydrogenated corn syrups are Lycasin, a commercially available productmanufactured by Roquette Corporation, and Hystar, a commerciallyavailable product manufactured by Lonza, Inc. The candy lozenges maycontain up to about 95% sorbitol, a mixture of sorbitol and mannitol ina ratio from about 9.5:0.5 up to about 7.5:2.5, and hydrogenated cornsyrup up to about 55%, by weight of the solid syrup component.

Boiled candy lozenges may be routinely prepared by conventional methodssuch as those involving fire cookers, vacuum cookers, andscraped-surface cookers also referred to as high speed atmosphericcookers.

Fire cookers involve the traditional method of making a boiled candylozenge base. In this method, the desired quantity of carbohydratebulking agent is dissolved in water by heating the agent in a kettleuntil the bulking agent dissolves. Additional bulking agent may then beadded and cooking continued until a final temperature of 145° C. to 156°C. is achieved. The batch is then cooled and worked as a plastic-likemass to incorporate additives such as flavors, colorants and the like.

A high-speed atmospheric cooker uses a heat-exchanger surface whichinvolves spreading a film of candy on a heat exchange surface, the candyis heated to 165° C. to 170° C. in a few minutes. The candy is thenrapidly cooled to 100° C. to 120° C. and worked as a plastic-like massenabling incorporation of the additives, such as flavors, colorants andthe like.

In vacuum cookers, the carbohydrate bulking agent is boiled to 125° C.to 132° C., vacuum is applied and additional water is boiled off withoutextra heating. When cooking is complete, the mass is a semi-solid andhas a plastic-like consistency. At this point, flavors, colorants, andother additives are admixed in the mass by routine mechanical mixingoperations.

The optimum mixing required to uniformly mix the flavoring agents,coloring agents and other additives during conventional manufacturing ofboiled candy lozenges is determined by the time needed to obtain auniform distribution of the materials. Normally, mixing times of from 4to 10 minutes have been found to be acceptable.

Once the boiled candy lozenge has been properly tempered, it may be cutinto workable portions or formed into desired shapes. A variety offorming techniques may be utilized depending upon the shape and size ofthe final product desired. A general discussion of the composition andpreparation of hard confections may be found in H. A. Lieberman,Pharmaceutical Dosage Forms: Tablets, Volume 1 (1980), Marcel Dekker,Inc., New York, N.Y. at pages 339 to 469, which disclosure isincorporated herein by reference.

The apparatus useful in accordance with the present invention comprisescooking and mixing apparatus well known in the confectionerymanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

In contrast, compressed tablet confections contain particulate materialsand are formed into structures under pressure. These confectionsgenerally contain sugars in amounts up to about 95%, by weight of thecomposition, and typical tablet excipients such as binders andlubricants as well as flavoring agents, coloring agents and the like.

In addition to hard confectionery materials, the lozenges of the presentinvention may be made of soft confectionery materials such as thosecontained in nougat. The preparation of soft confections, such asnougat, involves conventional methods, such as the combination of twoprimary components, namely (1) a high boiling syrup such as a cornsyrup, hydrogenated starch hydrolysate or the like, and (2) a relativelylight textured frappe, generally prepared from egg albumin, gelatin,vegetable proteins, such as soy derived compounds, sugarless milkderived compounds such as milk proteins, and mixtures thereof. Thefrappe is generally relatively light, and may, for example, range indensity from about 0.5 to about 0.7 grams/cc.

The high boiling syrup, or "bob syrup" of the soft confectionery isrelatively viscous and has a higher density than the frappe component,and frequently contains a substantial amount of carbohydrate bulkingagent such as a hydrogenated starch hydrolysate. Conventionally, thefinal nougat composition is prepared by the addition of the "bob syrup"to the frappe under agitation, to form the basic nougat mixture. Furtheringredients such as flavoring agents, additional carbohydrate bulkingagent, coloring agents, preservatives, medicaments, mixtures thereof andthe like may be added thereafter also under agitation. A generaldiscussion of the composition and preparation of nougat confections maybe found in B. W. Minifie, Chocolate. Cocoa and Confectionery: Scienceand Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn.(1980), at pages 424-425, which disclosure is incorporated herein byreference.

The procedure for preparing the soft confectionery involves knownprocedures. In general, the frappe component is prepared first andthereafter the syrup component is slowly added under agitation at atemperature of at least about 65° C., and preferably at least about 100°C. The mixture of components is continued to be mixed to form a uniformmixture, after which the mixture is cooled to a temperature below 80°C., at which point, the flavoring agent may be added. The mixture isfurther mixed for an additional period until it is ready to be removedand formed into suitable confectionery shapes.

The ingestible therapeutic wound healing compositions may also be in theform of a pharmaceutical suspension. Pharmaceutical suspensions of thisinvention may be prepared by conventional methods long established inthe art of pharmaceutical compounding. Suspensions may contain adjunctmaterials employed in formulating the suspensions of the art. Thesuspensions of the present invention can comprise:

(a) preservatives such as butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), benzoic acid, ascorbic acid, methyl paraben,propyl paraben, tocopherols, and the like, and mixtures thereof.Preservatives are generally present in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(b) buffers such as citric acid-sodium citrate, phosphoric acid-sodiumphosphate, and acetic acid-sodium acetate in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(c) suspending agents or thickeners such as cellulosics likemethylcellulose, carrageenans like alginic acid and its derivatives,xanthan gums, gelatin, acacis, and microcrystalline cellulose in amountsup to about 20%, and preferably from about 1% to about 15%, by weight ofthe suspension;

(d) antifoaming agents such as dimethyl polysiloxane in amounts up toabout 0.2%, and preferably from about 0.01% to about 0.1%, by weight ofthe suspension;

(e) sweetening agents such as those sweeteners well known in the art,including both natural and artificial sweeteners. Sweetening agents suchas monosaccharides, disaccharides and polysaccharides such as xylose,ribose, glucose (dextrose), mannose, galactose, fructose (levulose),sucrose (sugar), maltose, invert sugar (a mixture of fructose andglucose derived from sucrose), partially hydrolyzed starch, corn syrupsolids, dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenated starchhydrolysates and mixtures thereof may be utilized in amounts up to about60%, and preferably from about 20% to about 50%, by weight of thesuspension. Water-soluble artificial sweeteners such as solublesaccharin salts, i.e., sodium or calcium saccharin salts, cyclamatesalts, the sodium, ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like may beutilized in amounts from about 0.001% to about 5%, by weight of thesuspension;

(f) flavoring agents such as those flavors well known to the skilledartisan, such as natural and artificial flavors and mints, such aspeppermint, menthol, citrus flavors such as orange and lemon, artificialvanilla, cinnamon, various fruit flavors, both individual and mixed andthe like may be utilized in amounts from about 0.5% to about 5%, byweight of the suspension;

(g) coloring agents such as pigments which may be incorporated inamounts up to about 6%, by weight of the suspension. A preferredpigment, titanium dioxide, may be incorporated in amounts up to about2%, and preferably less than about 1%, by weight of the suspension. Thecoloring agents may also include natural food colors and dyes suitablefor food, drug and cosmetic applications. These colorants are known asF.D.& C. dyes and lakes. The materials acceptable for the foregoing usesare preferably water-soluble. Such dyes are generally present in amountsup to about 0.25%, and preferably from about 0.05% to about 0.2%, byweight of the suspension;

(h) decolorizing agents such as sodium metabisulfite, ascorbic acid andthe like may be incorporated into the suspension to prevent colorchanges due to aging. In general, decolorizing agents may be used inamounts up to about 0.25%, and preferably from about 0.05% to about0.2%, by weight of the suspension; and

(i) solubilizers such as alcohol, propylene glycol, polyethylene glycol,and the like may be used to solubilize the flavoring agents. In general,solubilizing agents may be used in amounts up to about 10%, andpreferably from about 2% to about 5%, by weight of the suspension.

The pharmaceutical suspensions of the present invention may be preparedas follows:

(A) admix the thickener with water heated from about 40° C. to about 95°C., preferably from about 40° C. to about 70° C., to form a dispersionif the thickener is not water soluble or a solution if the thickener iswater soluble;

(B) admix the sweetening agent with water to form a solution;

(C) admix the therapeutic wound healing composition with thethickener-water admixture to form a uniform thickener-therapeutic woundhealing composition;

(D) combine the sweetener solution with the thickener-therapeutic woundhealing composition and mix until uniform; and

(E) admix the optional adjunct materials such as coloring agents,flavoring agents, decolorants, solubilizers, antifoaming agents, buffersand additional water with the mixture of step (D) to form thesuspension.

The ingestible therapeutic wound healing compositions of this inventionmay also be in chewable form. To achieve acceptable stability andquality as well as good taste and mouth feel in a chewable formulationseveral considerations are important. These considerations include theamount of active substance per tablet, the flavoring agent employed, thedegree of compressibility of the tablet and the organoleptic propertiesof the composition.

Chewable therapeutic candy is prepared by procedures similar to thoseused to make soft confectionery. In a typical procedure, a boiledsugar-corn syrup blend is formed to which is added a frappe mixture. Theboiled sugar-corn syrup blend may be prepared from sugar and corn syrupblended in parts by weight ratio of about 90:10 to about 10:90. Thesugar-corn syrup blend is heated to temperatures above about 120° C. toremove water and to form a molten mass.

The frappe is generally prepared from gelatin, egg albumin, milkproteins such as casein, and vegetable proteins such as soy protein, andthe like, which is added to a gelatin solution and rapidly mixed atambient temperature to form an aerated sponge like mass. The frappe isthen added to the molten candy mass and mixed until homogeneous attemperatures between about 65° C. and about 120° C.

The ingestible therapeutic wound healing composition of the instantinvention can then be added to the homogeneous mixture as thetemperature is lowered to about 65° C.-95° C. whereupon additionalingredients can then be added such as flavoring agents and coloringagents. The formulation is further cooled and formed into pieces ofdesired dimensions.

A general discussion of the lozenge and chewable tablet forms ofconfectionery may be found in H. A. Lieberman and L. Lachman,Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, Inc., NewYork, N.Y. at pages 289 to 466, which disclosure is incorporated hereinby reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the hard and soft confectionery products. These amountsare readily determined by those skilled in the art without the need forundue experimentation.

In a preferred embodiment, the ingestible therapeutic wound healingcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and an ingestible vehicle, thatis a pharmaceutically acceptable carrier, in a quantity sufficient tobring the total amount of composition to 100%, by weight the ingestibletherapeutic wound healing composition. In a more preferred embodiment,the ingestible composition will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the ingestible composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and an ingestible vehicle in a quantity sufficient to bringthe total amount of composition to 100%, by weight the ingestibletherapeutic wound healing composition.

The present invention extends to methods of making the ingestibletherapeutic wound healing compositions. In such methods, an ingestibletherapeutic wound healing composition is prepared by admixing atherapeutically effective amount of the therapeutic wound healingcomposition with a pharmaceutically-acceptable carrier. The apparatususeful in accordance with the present invention comprises mixing andheating apparatus well known in the confectionery arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.The final ingestible therapeutic wound healing compositions are readilyprepared using methods generally known in the confectionery arts.

The therapeutic wound healing compositions may also be incorporated intochewing gums. In this form of the invention, the chewing gum compositioncontains a gum base, a bulking agent, the inventive therapeutic woundhealing composition, and various additives.

The gum base employed will vary greatly depending upon various factorssuch as the type of base desired, the consistency of gum desired and theother components used in the composition to make the final chewing gumproduct.

The gum base may be any water-insoluble gum base known in the art, andincludes those gum bases utilized for chewing gums and bubble gums.Illustrative examples of suitable polymers in gum bases include bothnatural and synthetic elastomers and rubbers. For example, thosepolymers which are suitable as gum bases include, without limitation,substances of vegetable origin such as chicle, crown gum, nispero,rosadinha, jelutong, perillo, niger gutta, tunu, balata, gutta-percha,lechicapsi, sorva, gutta kay, mixtures thereof and the like. Syntheticelastomers such as butadiene-styrene copolymers, polyisobutylene,isobutylene-isoprene copolymers, polyethylene, mixtures thereof and thelike are particularly useful.

The gum base may include a non-toxic vinyl polymer, such as polyvinylacetate and its partial hydrolysate, polyvinyl alcohol, and mixturesthereof. When utilized, the molecular weight of the vinyl polymer mayrange from about 2,000 up to and including about 94,000.

The amount of gum base employed will vary greatly depending upon variousfactors such as the type of base used, the consistency of the gumdesired and the other components used in the composition to make thefinal chewing gum product. In general, the gum base will be present inamounts from about 5% to about 94%, by weight of the final chewing gumcomposition, and preferably in amounts from about 15% to about 45%, andmore preferably in amounts from about 15% to about 35%, and mostpreferably in amounts from about 20% to about 30%, by weight of thefinal chewing gum composition.

The gum base composition may contain conventional elastomer solvents toaid in softening the elastomer base component. Such elastomer solventsmay comprise terpinene resins such as polymers of α-pinene or β-pinene,methyl, glycerol or pentaerythritol esters of rosins or modified rosinsand gums, such as hydrogenated, dimerized or polymerized rosins ormixtures thereof. Examples of elastomer solvents suitable for use hereininclude the pentaerythritol ester of partially hydrogenated wood or gumrosin, the pentaerythritol ester of wood or gum rosin, the glycerolester of wood rosin, the glycerol ester of partially dimerized wood orgum rosin, the glycerol ester of polymerized wood or gum rosin, theglycerol ester of tall oil rosin, the glycerol ester of wood or gumrosin and the partially hydrogenated wood or gum rosin and the partiallyhydrogenated methyl ester of wood or rosin, mixtures thereof, and thelike. The elastomer solvent may be employed in amounts from about 5% toabout 75%, by weight of the gum base, and preferably from about 45% toabout 70%, by weight of the gum base.

A variety of traditional ingredients may be included in the gum base ineffective amounts such as plasticizers or softeners such as lanolin,palmitic acid, oleic acid, stearic acid, sodium stearate, potassiumstearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate,propylene glycol monostearate, acetylated monoglyceride, glycerine,mixtures thereof, and the like may also be incorporated into the gumbase to obtain a variety of desirable textures and consistencyproperties. Waxes, for example, natural and synthetic waxes,hydrogenated vegetable oils, petroleum waxes such as polyurethane waxes,polyethylene waxes, paraffin waxes, microcrystalline waxes, fatty waxes,sorbitan monostearate, tallow, propylene glycol, mixtures thereof, andthe like may also be incorporated into the gum base to obtain a varietyof desirable textures and consistency properties. These traditionaladditional materials are generally employed in amounts up to about 30%,by weight of the gum base, and preferably in amounts from about 3% toabout 20%, by weight of the gum base.

The gum base may include effective amounts of mineral adjuvants such ascalcium carbonate, magnesium carbonate, alumina, aluminum hydroxide,aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate andthe like as well as mixtures thereof. These mineral adjuvants may serveas fillers and textural agents. These fillers or adjuvants may be usedin the gum base in various amounts. Preferably the amount of filler whenused will be present in an amount up to about 60%, by weight of thechewing gum base.

The chewing gum base may additionally include the conventional additivesof coloring agents, antioxidants, preservatives and the like. Forexample, titanium dioxide and other dyes suitable for food, drug andcosmetic applications, known as F.D. & C. dyes, may be utilized. Anantioxidant such as butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), propyl gallate, and mixtures thereof, may also beincluded. Other conventional chewing gum additives known to one havingordinary skill in the chewing gum art may also be used in the chewinggum base.

The gum composition may include effective amounts of conventionaladditives selected from the group consisting of sweetening agents(sweeteners), plasticizers, softeners, emulsifiers, waxes, fillers,bulking agents, mineral adjuvants, flavoring agents (flavors,flavorings), coloring agents (colorants, colorings), antioxidants,acidulants, thickeners, mixtures thereof and the like. Some of theseadditives may serve more than one purpose. For example, in sugarless gumcompositions, the sweetener, e.g., sorbitol or other sugar alcohol ormixtures thereof, may also function as a bulking agent. Similarly, insugar containing gum compositions, the sugar sweetener can also functionas a bulking agent.

The plasticizers, softeners, mineral adjuvants, colorants, waxes andantioxidants discussed above as being suitable for use in the gum basemay also be used in the gum composition. Examples of other conventionaladditives which may be used include emulsifiers, such as lecithin andglyceryl monostearate, thickeners, used alone or in combination withother softeners, such as methyl cellulose, alginates, carrageenan,xanthan gum, gelatin, carob, tragacanth, locust bean, and carboxy methylcellulose, acidulants such as malic acid, adipic acid, citric acid,tartaric acid, fumaric acid, and mixtures thereof, and fillers, such asthose discussed above under the category of mineral adjuvants. Thefillers when used may be utilized in an amount up to about 60%, byweight of the gum composition.

Bulking agents (carriers, extenders) suitable for use in chewing gumsinclude sweetening agents selected from the group consisting ofmonosaccharides, disaccharides, poly-saccharides, sugar alcohols, andmixtures thereof; polydextrose; maltodextrins; minerals, such as calciumcarbonate, talc, titanium dioxide, dicalcium phosphate, and the like.Bulking agents may be used in amounts up to about 90%, by weight of thefinal gum composition, with amounts from about 40% to about 70%, byweight of the gum composition being preferred, with from about 50% toabout 65%, by weight, being more preferred and from about 55% to about60%, by weight of the chewing gum composition, being most preferred.

The sweetening agent used may be selected from a wide range of materialsincluding water-soluble sweeteners, water-soluble artificial sweeteners,water-soluble sweeteners derived from naturally occurring water-solublesweeteners, dipeptide based sweeteners, and protein based sweeteners,including mixtures thereof. Without being limited to particularsweeteners, representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribulose, glucose(dextrose), mannose, galactose, fructose (levulose), sucrose (sugar),maltose, invert sugar (a mixture of fructose and glucose derived fromsucrose), partially hydrolyzed starch, corn syrup solids,dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenated starchhydrolysates and mixtures thereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspartame)and materials described in U.S. Pat. No. 3,492,131,L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amide hydrate(Alitame), methyl esters of L-aspartyl-L-phenylglycerine andL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), known, for example, under the product designation ofSucralose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand II).

In general, an effective amount of sweetener is utilized to provide thelevel of bulk and/or sweetness desired, and this amount will vary withthe sweetener selected. This amount of sweetener will normally bepresent in amounts from about 0.0025% to about 90%, by weight of the gumcomposition, depending upon the sweetener used. The exact range ofamounts for each type of sweetener is well known in the art and is notthe subject of the present invention. The amount of sweetener ordinarilynecessary to achieve the desired level of sweetness is independent fromthe flavor level achieved from flavor oils.

Preferred sugar based-sweeteners are sugar (sucrose), corn syrup andmixtures thereof. Preferred sugarless sweeteners are the sugar alcohols,artificial sweeteners, dipeptide based sweeteners and mixtures thereof.Preferably, sugar alcohols are used in the sugarless compositionsbecause these sweeteners can be used in amounts which are sufficient toprovide bulk as well as the desired level of sweetness. Preferred sugaralcohols are selected from the group consisting of sorbitol, xylitol,maltitol, mannitol, and mixtures thereof. More preferably, sorbitol or amixture of sorbitol and mannitol is utilized. The gamma form of sorbitolis preferred. An artificial sweetener or dipeptide based sweetener ispreferably added to the gum compositions which contain sugar alcohols.

The coloring agents useful in the gum compositions are used in amountseffective to produce the desired color. These coloring agents includepigments which may be incorporated in amounts up to about 6% by weightof the gum composition. A preferred pigment, titanium dioxide, may beincorporated in amounts up to about 2%, and preferably less than about1% by weight of the composition. The colorants may also include naturalfood colors and dyes suitable for food, drug and cosmetic applications.These colorants are known as F.D.& C. dyes and lakes. The materialsacceptable for the foregoing uses are preferably water-soluble.Illustrative nonlimiting examples include the indigoid dye known asF.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo.1 comprises a triphenylmethane dye and is the monosodium salt of 4-4-(N-ethyl-p-sulfoniumbenzylamino) diphenylmethylene!-1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5cyclohexadieneimine!. A fullrecitation of all F.D.& C. colorants and their corresponding chemicalstructures may be found in the Kirk-Othmer Encyclopedia of ChemicalTechnology, 3rd Edition, in volume 5 at pages 857-884, which text isincorporated herein by reference.

Suitable oils and fats usable in gum compositions include partiallyhydrogenated vegetable or animal fats, such as coconut oil, palm kerneloil, beef tallow, lard, and the like. These ingredients when used aregenerally present in amounts up to about 7%, by weight, and preferablyup to about 3.5%, by weight of the gum composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into a chewing gum. These amounts are readily determined bythose skilled in the art without the need for undue experimentation. Ina preferred embodiment, the final chewing gum composition will comprisethe therapeutic wound healing composition in an amount from about 0.1%to about 10% and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition. In a more preferred embodiment, the final chewing gumcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the final chewing gum composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition.

The present invention extends to methods of making the therapeuticchewing gum compositions. The therapeutic wound healing compositions maybe incorporated into an otherwise conventional chewing gum compositionusing standard techniques and equipment known to those skilled in theart. The apparatus useful in accordance with the present inventioncomprises mixing and heating apparatus well known in the chewing gummanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

For example, a gum base is heated to a temperature sufficiently highenough to soften the base without adversely effecting the physical andchemical make up of the base. The optimum temperatures utilized may varydepending upon the composition of the gum base used, but suchtemperatures are readily determined by those skilled in the art withoutundue experimentation.

The gum base is conventionally melted at temperatures that range fromabout 60° C. to about 120° C. for a period of time sufficient to renderthe base molten. For example, the gum base may be heated under theseconditions for a period of about thirty minutes just prior to beingadmixed incrementally with the remaining ingredients of the base such asthe plasticizer, fillers, the bulking agent and/or sweeteners, thesoftener and coloring agents to plasticize the blend as well as tomodulate the hardness, viscoelasticity and formability of the base. Thechewing gum base is then blended with the therapeutic wound healingcomposition of the present invention which may have been previouslyblended with other traditional ingredients. Mixing is continued until auniform mixture of gum composition is obtained. Thereafter the gumcomposition mixture may be formed into desirable chewing gum shapes.

In a specific embodiment, the invention is directed to a therapeuticpharmaceutical composition for preventing and reducing injury tomammalian cells, and increasing the resuscitation rate of injuredmammalian cells, which comprises:

(A) a therapeutically effective amount of a therapeutic wound healingcomposition of Embodiment One (I) selected from the group consisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(4) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be selected from the groupconsisting of pharmaceutical appliances, topical vehicles, andingestible vehicle.

In another specific embodiment, the invention is directed to a methodfor preparing a therapeutic pharmaceutical composition for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of:

(A) providing a therapeutically effective amount of a therapeutic woundhealing composition of Embodiment One (I) selected from the groupconsisting of:

(I.A)

(1) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)

(2) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)

(3) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)

(4) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the therapeutic wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form atherapeutic pharmaceutical composition.

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

The present invention is further illustrated by the following exampleswhich are not intended to limit the effective scope of the claims. Allparts and percentages in the examples and throughout the specificationand claims are by weight of the final composition unless otherwisespecified.

E. Examples Of The Wound Healing Compositions Of Embodiment One (I.A-D)

Study 1

This study demonstrates a comparison of the viability of U937 monocyticcells after exposure of the cells to various antioxidants andcombinations of antioxidants. This study also demonstrate a comparisonof the levels of hydrogen peroxide produced by U937 monocytic cells andmammalian epidermal keratinocytes after exposure of the cells to variousantioxidants and combinations of antioxidants. The results of this studyare illustrated in FIGS. 1-4 and examples 1-26 below.

Mammalian epidermal keratinocytes and monocytes were employed to examinethe ability of various antioxidants to reduce levels of hydrogenperoxide in these cells. Hydrogen peroxide was measured after the cellswere exposed to ultraviolet light in the wavelength range from 290 to320 nm (UV-B) or to the inflammatory compound12-0-tetradecanoyl-phorbol-13-acetate (TPA). Sodium pyruvate was testedat various concentrations to determine the effect of concentrations ofthis antioxidant on the hydrogen peroxide production by epidermal cellsand monocytes. Magnesium pyruvate, calcium pyruvate, zinc pyruvate, andcombinations of sodium pyruvate with ascorbic acid, lactic acid, andVitamin E were then tested to determine the effect of these salts andcombinations of antioxidants on the hydrogen peroxide production byepidermal cells and monocytes.

Mammalian epidermal keratinocytes were isolated by trypsinization ofepithelial sheets and grown in modified basal MCDB 153 mediumsupplemented with epidermal growth factor, bovine pituitary extract, andhydrocortisone. Cells were maintained in a humidified incubator with 5%carbon dioxide at 37° C.

Keratinocytes were seeded in 60 mm culture dishes at a cell density of3×105 cells per dish and the cultures were exposed to 1 M.E.D. dose ofultraviolet-B light (100 mJ/cm²) or treated with 100 ng/ml of TPA.

U937 monocytic cells are a cultured cell line grown in RPMI media with10% fetal calf serum. Cells were maintained in a 60 mm culture dish at5% carbon dioxide at 37° C. at a seeding density not exceeding 1×106cells per dish.

Sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E weredissolved in distilled water, with sufficient surfactant. Theconcentrations of the sodium pyruvate solutions prepared were 1 mM, 10mM, 50 mM, 100 mM, and 200 mM. The concentrations of the lactic acidsolutions prepared were 1.0%, 0.1%, and 0.05%. The concentrations of theascorbic acid solutions prepared were 1.0%, 0.1%, 0.05%, and 0.025%. Theconcentrations of the Vitamin E solutions prepared were 1 U, 10 U, 50 U,and 100 U. The test solutions were adjusted to a pH value of 7.4 with1.0N sodium hydroxide solution and then sterile filtered. Theappropriate concentration of test solution or combination of testsolutions was added to the cells immediately prior to exposure of thecells to ultraviolet light-B or TPA 100 ng/ml!. Stock solutions wereprepared so that the vehicle did not constitute more than 1% of thetotal volume of the culture media.

Intracellular hydrogen peroxide production by mammalian epidermalkeratinocytes and U937 monocytes was measured using dichlorofluoresceindiacetate (DCFH-DA, Molecular Probes, Eugene, Oreg.). DCFH-DA is anon-polar non-fluorescent compound that readily diffuses into cellswhere it is hydrolyzed to the polar non-fluorescent derivative DCFHwhich then becomes trapped within the cells. In the presence ofintracellular hydrogen peroxide, DCFH is oxidized to the highlyfluorescent compound DCF. Hence, cellular fluorescence intensity isdirectly proportional to the level of intracellular hydrogen peroxideproduced. Cellular fluorescence intensity can be monitored byfluorimetry and by flow cytometry.

Mammalian epidermal keratinocytes and U937 cultured monocytes (1×106 perdish) were incubated at 37° C. with 5 uM of DCFH-DA. Production ofhydrogen peroxide was measured using a Coulter Profile analytical flowcytometer. Linear and log intensity of green fluorescence data wascollected. For each analysis, a quantity of 10,000 to 20,000 events wasaccumulated. Optical alignment for the instrument was performed daily.Coefficients of variation for forward angle light scatter and integratedgreen fluorescence were generally less than two. Each analysis wasrepeated three times and the quantitation of fluorescence was expressedin terms of femtomoles (fmol, 10⁻¹⁵ moles) of DCF oxidized per cell,which is a direct measure of the intracellular hydrogen peroxideproduced. Alternatively, in the saturated and unsaturated fatty acidexamples in examples 27-52, fluorimetry was used to assess the DCFoxidation per cell.

The viability of the U937 monocytic cells after exposure of the cells tovarious antioxidants for 24 hours was measured. The viability of thecells was determined by exposing the cells to the dye propidium iodide.Permeable cell membranes which absorbed the dye were not consideredviable. The viability of the cells was represented as the percentage ofcells that excluded propidium iodide.

FIG. 1 depicts in bar graph format the viability of U937 monocytic cellsafter exposure of the cells to no antioxidant (Example 1, control), tosodium pyruvate (Example 2), to ascorbic acid (Example 3), to lacticacid (Example 4), and to Vitamin E (Example 5). FIG. 2 depicts in bargraph format the viability of U937 monocytic cells after exposure of thecells to various combinations of antioxidants. Specifically, theviability of U937 monocytic cells was measured after exposure to noantioxidant (Example 6, control), to ascorbic acid and lactic acid(Example 7), to ascorbic acid and Vitamin E (Example 8), to sodiumpyruvate and ascorbic acid (Example 9), to sodium pyruvate and lacticacid (Example 10), to sodium pyruvate and Vitamin E (Example 11), tolactic acid and Vitamin E (Example 12), and to sodium pyruvate, ascorbicacid, and lactic acid (Example 13).

FIG. 1 shows that ascorbic acid is cytotoxic to monocytes atconcentrations as low as 0.25%. FIG. 2 shows that the cytotoxicity ofascorbic acid was reversed by the addition of 10 mM of sodium pyruvate.FIGS. 1 and 2 show that the viability rate of 15% to 20% of the cellswhen treated with ascorbic acid was increased to 95% to 98% uponaddition of sodium pyruvate. Lactic acid and Vitamin E did not reversethe cytotoxicity of ascorbic acid.

Sodium pyruvate was then tested at various concentrations to determinethe effect of concentrations of this antioxidant on the hydrogenperoxide production by epidermal cells and monocytes. Mammalianepidermal keratinocytes and monocytes were exposed to (a) 1 M.E.D. doseof ultraviolet light-B and (b) 100 ng/ml of12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of sodiumpyruvate at the following concentrations: 200 mM, 100 mM, 50 mM, 10 mM,1 mM.

The optimum concentration of sodium pyruvate to reduce the hydrogenperoxide production by epidermal cells and monocytes was found to be 10mM. Concentrations of sodium pyruvate of 50 mM and above were cytotoxicto both epidermal keratinocytes and monocytes.

Magnesium pyruvate, calcium pyruvate, zinc pyruvate, ascorbic acid,lactic acid, and Vitamin E, and combinations of sodium pyruvate withascorbic acid, lactic acid, and Vitamin E were then tested to determinethe effect of these salts and combinations of antioxidants on thehydrogen peroxide production by epidermal cells and monocytes. Thefollowing test solutions were prepared.

(a) sodium pyruvate 10 mM!;

(b) zinc salt 10 mM!;

(c) magnesium salt 10 mM!;

(d) calcium salt 10 mM!;

(e) sodium pyruvate 10 mM! and ascorbic acid 0.025%!;

(f) sodium pyruvate 10 mM! and lactic acid 0.05%!;

(g) sodium pyruvate 10 mM!, lactic acid, 0.05%!, and ascorbic acid0.025%!;

(h) lactic acid 1.0%, 0.1%, and 0.05%!;

(i) ascorbic acid 1.0%, 0.1%, 0.05%, and 0.025%!;

(j) Vitamin E 1 U, 10 U, 50 U, and 100 U!; and

(k) vehicle solvent controls.

There was no significant difference among the zinc, magnesium, andcalcium salts of pyruvic acid on the hydrogen peroxide production byepidermal cells and monocytes. The zinc and calcium salts of pyruvicacid induced differentiation of keratinocytes. For convenience, thesodium salt was used in subsequent tests.

The optimum concentration of lactic acid to reduce the hydrogen peroxideproduction by epidermal cells and monocytes was found to be 0.05%. Theoptimum concentration of ascorbic acid was found to be 0.025%. Thehigher concentrations of both of these compounds were found to becytotoxic to both types of cells. The optimum concentration of Vitamin Ewas found to be 50 U.

FIG. 3 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to noantioxidant (Example 14, control), to sodium pyruvate (Example 15), toascorbic acid (Example 16), to lactic acid (Example 17), and to VitaminE (Example 18). Sodium pyruvate and Vitamin E significantly reduced thehydrogen peroxide production by monocytes.

FIG. 4 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to variouscombinations of antioxidants. Specifically, the levels of hydrogenperoxide produced by U937 monocytic cells were measured after exposureto no antioxidant (Example 19, control), to ascorbic acid and lacticacid (Example 20), to ascorbic acid and Vitamin E (Example 21), tosodium pyruvate and ascorbic acid (Example 22), to sodium pyruvate andlactic acid (Example 23), to sodium pyruvate and Vitamin E (Example 24),to lactic acid and Vitamin E (Example 25), and to sodium pyruvate,ascorbic acid, and lactic acid (Example 26). The combination of lacticacid (0.05%) and Vitamin E (50 U) significantly reduced the hydrogenperoxide production by monocytes.

The morphological alterations in epidermal keratinocytes were observedin control cultures and in cultures exposed to ultraviolet-B. Cells inthe layer closest to the dermis are basal keratinocytes. These cellsproliferate and migrate into the spinous and granular layers of theepidermis where the cells begin to differentiate. The differentiationpattern results in cells enucleating and forming cornified envelopes atthe uppermost portion of the epidermis, the statum corneum. Thedifferentiation of keratinocytes is controlled by the levels of calcium,magnesium, and other elements in the medium. Cells in culture systemspromoting differentiation appear as an epidermal sheet formingattachments or tight junctions with each other. Keratinocytes thatbecome nonadherent or float in the media were considered responding to acytotoxic event.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the following control cultures:

10 mM Sodium Pyruvate: Tight junctions of cells were formed and theproliferation rate of the cells was higher than the rate of the controlcells.

0.025% Ascorbic Acid: Cells were floating in a cytotoxic response toascorbic acid.

0.025% Ascorbic acid and 10 mM Sodium Pyruvate: Few tight junctions ofcells were observed and cells appeared similar to the cells in thesodium pyruvate culture.

0.05% Lactic Acid: Cells appeared dramatically altered as an epidermalsheet and as flat granular cells.

0.05% Lactic Acid and 10 mM Sodium Pyruvate: Cells formed an epidermalsheet but appeared smaller than the cell in the lactic acid culture.

50 U Vitamin E: Cells appeared the same as the cells in the controlculture.

50 U Vitamin E and 10 mM Sodium Pyruvate: Cells increased in number andchanged in appearance resembling the cells in the sodium pyruvateculture.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the corresponding cultures exposed toultraviolet light-B, 100 mJoules, for 24 hours:

10 mM Sodium Pyruvate: Cells proliferated more rapidly than the cells inthe control culture.

0.025% Ascorbic Acid: Cells were nonadherent and floating in a cytotoxicresponse to ascorbic acid greater than the cytotoxic response of thecorresponding cells without ultraviolet-B light exposure.

0.05% Lactic Acid: Cells formed an epidermal sheet and were moregranular than cells in the control culture without ultraviolet-B lightexposure.

50 U Vitamin E: Cell growth was inhibited but cells appeared similar tocells in the control culture without ultraviolet-B light exposure.

50 U Vitamin E and 10 mM Sodium Pyruvate: Cells appeared similar tocells in the control culture and proliferated to a greater extent thancells in the control cultures without ultraviolet-B light exposure.

Morphological alterations in the U937 monocytic cell line were alsoobserved for control cultures and cultures exposed to ultravioletlight-B, 100 mJoules, for 24 hours. The following compounds andcombination of compounds, at the concentrations set out below,significantly inhibited the levels of hydrogen peroxide produced by U937monocytic cells

Sodium pyruvate at 10 mM and 50 mM;

Vitamin E at 50 U and 100 U; and

Lactic acid at 0.05% and Vitamin E at 50 U.

Examples Of The Wound Healing Compositions Of Embodiment One (I.A-D)

Study 2

This study demonstrates a comparison of the levels of hydrogen peroxideproduced by U937 monocytic cells and epidermal keratinocytes afterexposure of the cells to various combinations of antioxidants with andwithout a mixture of saturated and unsaturated fatty acids. The resultsof this study are illustrated in FIGS. 5-7 and examples 27-52 below.

Mammalian epidermal keratinocytes and U937 monocytic cells and the testsolutions of sodium pyruvate, lactic acid, ascorbic acid, and Vitamin Ewere prepared as describe above for Examples 1-26. Intracellularhydrogen peroxide production by the mammalian epidermal keratinocytesand U937 monocytes was also measured as described above.

A mixture of fatty acids derived from chicken fat was prepared foraddition to the cultured cells by mixing 0.1% of the chicken fat withthe culture media. At the temperature of the culture media, 37° C., thechicken fat was miscible. This chicken fat mixture was added to culturesof cells prior to exposure of the cells to ultraviolet-B light or TPAtreatment.

As set out in examples 1-26, mammalian epidermal keratinocytes andmonocytes were exposed to (a) 1 M.E.D. dose of ultraviolet light-B and(b) 100 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in the presence ofvarious antioxidants and combinations of antioxidants with and without amixture of saturated and unsaturated fatty acids 0.1%, 0.5%, and 1.0%chicken fat!.

FIG. 5 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to variouscombinations of antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by U937 monocytic cells were measured after exposure to lacticacid and Vitamin E without fatty acids (Example 27) and with fatty acids(Example 28), to ascorbic acid and lactic acid without fatty acids(Example 29) and with fatty acids (Example 30), and to ascorbic acid andVitamin E without fatty acids (Example 31) and with fatty acids (Example32). The ability of the combinations of lactic acid and Vitamin E,ascorbic acid and lactic acid, and ascorbic acid and Vitamin E to reducethe hydrogen peroxide production by monocytes was increased in thepresence of fatty acids. The most effective combination to reduce thehydrogen peroxide production of monocytes was lactic acid (0.05%) andVitamin E (50 E) in the presence of a mixture of saturated andunsaturated fatty acids (0.5 %).

FIG. 6 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by epidermal keratinocytes were measured after exposure to noantioxidant without fatty acids (Example 33, control) and with fattyacids (Example 34), to sodium pyruvate without fatty acids (Example 35)and with fatty acids (Example 36), to ascorbic acid without fatty acids(Example 37) and with fatty acids (Example 38), to lactic acid withoutfatty acids (Example 39) and with fatty acids (Example 40), and toVitamin E without fatty acids (Example 41) and with fatty acids (Example42). The ability of sodium pyruvate and Vitamin E to reduce the hydrogenperoxide production by epidermal keratinocytes was increased in thepresence of fatty acids. The most effective combinations to reduce thehydrogen peroxide production of epidermal keratinocytes were sodiumpyruvate in combination with a mixture saturated and unsaturated fattyacids and Vitamin E in combination with a mixture of saturated andunsaturated fatty acids.

FIG. 7 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids. Specifically, the levels ofhydrogen peroxide produced by epidermal keratinocytes were measuredafter exposure to no antioxidant without fatty acids (Example 43,control) and with fatty acids (Example 44), to sodium pyruvate andascorbic acid without fatty acids (Example 45) and with fatty acids(Example 46), to sodium pyruvate and lactic acid without fatty acids(Example 47) and with fatty acids (Example 48), to sodium pyruvate andVitamin E without fatty acids (Example 49) and with fatty acids (Example50), and to ascorbic acid and Vitamin E without fatty acids (Example 51)and with fatty acids (Example 52). The ability of all combinations ofantioxidants to reduce the hydrogen peroxide production by epidermalkeratinocytes was increased in the presence of fatty acids. In order ofpotency, the most effective combinations to reduce the hydrogen peroxideproduction of epidermal keratinocytes were sodium pyruvate and VitaminE, sodium pyruvate and lactic acid, and Vitamin E, each in combinationwith a mixture of saturated and unsaturated fatty acids (0.5%).

Because of the cytotoxicity of cells towards ascorbic acid describedabove, the ascorbic acid combinations without sodium pyruvate were notconsidered significantly different from the control test solution.

Summary Analysis Of The Data From Studies 1 and 2

Human epidermal keratinocytes were isolated by trypsinization ofepithelial sheets and grown in modified base MCDB 153 mediumsupplemented with epidermal growth factor and bovine pituitary extract.Cells were seeded in culture dishes at a density of 3×10⁵ /dish. Priorto exposure to UV B light (100 mJ/cm²) or treatment with 100 ng/ml TPA,the cultures were treated with the appropriate concentration of woundhealing components. Intracellular production of hydrogen peroxide wasmeasured using DCFH-DA, a nonpolar compound that readiiy diffuses intocells, hydrolyzad to a nonpolar derivative. In the presence ofintracellular hydrogen peroxide, DCFH is oxidized to a highlyfluorescent compound DCF. Thus, cellular fluorescence intensity isdirectly proportional to levels of hydrogen peroxide produced and can bemonitored by flow cytometry. Hydrogen peroxide is cytotoxic, thereforelower levels of hydrogen peroxide production is desirable for cellularviability.

In all cases, the three component wound healing composition surpassedthe predicted outcomes, clearly demonstrating unpredicted synergy.

    ______________________________________    Results    1                  2        3      4    ______________________________________    1 -   Control          250      250  0    2 -   Fatty Acids      250      230  -20          (0.5%)    3 -   Sodium Pyruvate  250      490  +240          (10 mM)    4 -   Vitamin E        250      400  +150          (50 units)    5 -   Pyruvate &       250      430  +180          Fatty Acids    6 -   Vitamin E &      250      200  -50          Fatty Acids    7 -   Pyruvate &       250      290  +40          Vitamin E    8 -   Pyruvate &       250      120  -130          Vitamin E & Fatty Acids          (wound healing composition)    ______________________________________     Column 1 shows the different treatment groups.     Column 2 shows the production of H.sub.2 O.sub.2 in control cells     (fmol/cell).     Column 3 shows the production of H.sub.2 O.sub.2 after treatment with     wound healing components.     Column 4 shows the difference in production of H.sub.2 O.sub.2 from     control after the treatment.

All comparisons were assessed against the controls, which produced 250H₂ O₂ fmol/cell. The positive numbers represent H₂ O₂ production inexcess of the control and the negative numbers represent H₂ O₂production below the control. These results are set out in FIG. 8.

    ______________________________________    Combination of Single Ingredient Effects    Fatty Acids (-20) & Vitamin E (+150) & Pyruvate (+240)    +370 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    500 Is The Difference Between Predicted Effect minus Actual effect    (Synergy)    Combination of Paired and Single Ingredients    Pyruvate & Fatty Acids (+180) & vitamin E (+150)    +330 Is The Predicted Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    460 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    Vitamin E & Fatty Acids (-50) & Pyruvate (+240)    +190 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    320 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    (Pyruvate & Vitamin E (+40) & Fatty Acids (-20)    +20 Is The Predicted Three Component Effect    -130 Is The Wound healing composition Actual Effect    150 Is The Difference between Predicted Effect minus Actual Effect    (Synergy)    ______________________________________

In all cases, the three component wound healing composition surpassedthe predicted outcomes clearly demonstrating unpredicted synergy.

Examples Of The Wound Healing Compositions Of Embodiment One (I.A-D)

Study 3

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic wound healing compositions of the present inventionversus conventional wound healing compositions. The results of thisstudy are illustrated in examples A-D.

The wound healing compositions of Examples A-D were prepared having thecompositions set out in Table A.

                  TABLE A    ______________________________________             Examples               A    Ingredient PREP.-H ™                          B        C      D    ______________________________________    sodium pyruvate               --         2%       --     --    vitamin E  --         1%       --     --    chicken fat               --         2%       --     --    LYCD       2000 U*    2400 U   2400 U --    shark liver oil               3%*        3%       3%     --    petrolatum in         64%      66.5%  68%    mineral oil               amounts    22.53%   25.03% 26.8%    paraffin   totaling   5%       5%     5%    emulsifier 100%*      0.2%     0.2%   0.2%    ______________________________________     *These components are present in PREPARATION H

Wound healing composition A was commercially available PREPARATION H™.Wound healing composition B was a petrolatum base formulation containinglive yeast cell derivative, shark oil, and a mixture of sodium pyruvate,vitamin E, and chicken fat. Wound healing composition C was a petrolatumbase formulation containing live yeast cell derivative and shark oil.Wound healing composition D was a petrolatum base formulation only.

Wound healing studies were carried out using hairless mice (SKR-1,Charles River) 6-8 weeks in age. One group of mice were untreated as acontrol group and were referred to as Example E. In each group therewere 6 mice for evaluation at either day 3 or day 7 for a total numberof 60 animals in the study. The mice were anesthetized with ether and amidline 3 cm full thickness longitudinal incision was made with a number10 scalpel blade. Incisions were closed using steel clips at 1 cmintervals. Formulations A-D set out above were applied in a randomizedblinded study to the wounds on day 0 at 2 hours following wounding andreapplied at 24 hour intervals during the 7 days of the study. Thewounds were examined daily and scored on a basis of 0-5 for closure oneach day of the study, with a score of 5 representing the wound besthealed.

The animals were sacrificed on day 3 and day 7 using cervicaldislocation. The dorsal skin including the incision was dissectedwithout the subcutaneous tissue. The skin was placed in neutral bufferedformalin and subsequently sectioned and stained with hematoxylin andeosin. The wounds were examined microscopically and representativetissue sections were photographed.

On each day of the experiment, the score and rank order of theformulations for closure of wounds and speed of healing were as follows:

    B(5)>>D(4)>>C(2)>/=E, Control (2)>A(1)

Photographs of the wounded mice on day 4 are set out in FIGS. 9A-9D and10.

FIGS. 9A-9D and 10 show that Formulation B, which was a petrolatum baseformulation containing live yeast cell derivative, shark oil, and amixture of sodium pyruvate, vitamin E, and chicken fat, was asignificantly better wound healing agent than the other formulations.These results are supported by the subjective grading of the woundclosures and the speed of healing on each day (1-7) of the experiment aswell as on the objective histological examination of tissue sections tomeasure the extent of inflammatory cell infiltrate within the wound andthe extent of epithelialization at the wound edges. The final result wasthat less scar tissue was present at day 7 on the mice treated withFormulation B.

Formulation D, which was a white petrolatum formulation only, was judgedto be significantly more effective to promote healing than eitherFormulation C, which was a petrolatum base formulation containing sharkliver oil and live yeast cell derivative, or Formulation A, which wasPREPARATION H™. The superior ability of Formulation D over Formulation Cto improve healing may result from a delay in the healing process causedwhen the live yeast cell derivative is depleted and the cells shift toan alternative nutrient source. The presence of the mixture of sodiumpyruvate, vitamin E, and chicken fat in Formulation B apparently offsetsthe depletion of the live yeast cell derivative.

Formulation C, which was a petrolatum base formulation containing liveyeast cell derivative and shark oil, was judged comparable to thecontrol (untreated wound) in speed of wound closure and extent ofhealing. Formulation A, which was PREPARATION H™, appeared to be theleast effective healing formulation by both subjective grading of woundhealing and by objective examination of tissue sections. The superiorability of Formulation D and Formulation C over Formulation A to improvehealing may be due to their ability to act as an occlusive wounddressing that prevents transepidermal water loss and thus promoteshealing and wound closure. The poor ability of Formulation A to improvehealing may be due to the potential cytotoxicity of phenylmercuricnitrate present in PREPARATION H™ as a preservative.

These results show that the wound healing compositions of the presentinvention which comprise a mixture of sodium pyruvate, vitamin E, andchicken fat increase the proliferation and resuscitation rate ofmammalian cells. The wound healing compositions mediate low levels ofoxygen in the initial stages of healing to suppress oxidative damage andhigher levels of oxygen in the later stages of healing to promotecollagen formation.

2. The Antiviral-Wound Healing Compositions

A. Embodiment Two (II.A-D+V)

As set out above, applicant has discovered therapeutic antiviral-woundhealing compositions (II.A-D+V) which comprise an antiviral agent (V)and the wound healing compositions of Embodiment One (I.A-D).Preferably, the wound healing composition (I.A) comprises (a) pyruvate,(b) an antioxidant, and (c) a mixture of saturated and unsaturated fattyacids. Applicant has found that the combination of an antiviral agentand a wound healing composition results in a therapeutic antiviral-woundhealing composition which reduces the size, duration, and severity oforal and vaginal wounds suffered from viruses such as herpes.

The antiviral agents in the antiviral-wound healing compositions of thepresent invention may be selected from a wide variety of water-solubleand water-insoluble drugs and their acid addition or metallic salts.Both organic and inorganic salts may be used provided the antiviralagent maintains its medicament value. The antiviral agents may beselected from a wide range of therapeutic agents and mixtures oftherapeutic agents which may be administered in sustained release orprolonged action form. Nonlimiting illustrative categories of suchantiviral agents include RNA synthesis inhibitors, protein synthesisinhibitors, immunostimulating agents, protease inhibitors, andcytokines. Nonlimiting illustrative specific examples of such antiviralagents include the following medicaments.

(a) Acyclovir (9- (2-hydroxyethyloxy)methyl!guanine, tradename--ZOVIRAX™) is an antiviral drug for oral administration. Acycloviris a white, crystalline powder with a molecular weight of 225 daltonsand a maximum solubility in water of 2.5 mg/mL at 37° C. Acyclovir is asynthetic purine nucleoside analogue with in vitro and in vivoinhibitory activity against human herpes viruses including herpessimplex types 1 (HSV-1) and 2 (HSV-2), varicella-zoster virus (VZV),Epstein-Barr virus (EBV), and cytomegalovirus (CMV).

(b) Foscarnet sodium (phosphonoformic acid trisodium salt, tradename--FOSCAVIR™) is an antiviral drug for intravenous administration.Foscarnet sodium is a white, crystalline powder containing 6 equivalentsof water of hydration with an empirical formula of Na₃ CO₆ P.6 H₂ O anda molecular weight of 300.1. Foscarnet sodium has the potential tochelate divalent metal ions such as calcium and magnesium, to formstable coordination compounds. Foscarnet sodium is an organic analogueof inorganic pyrophosphate that inhibits replication of all known herpesviruses in vitro including cytomegalovirus (CMV), herpes simplex virustypes 1 and 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Epstein-Barrvirus (EBV), and varicella-zoster virus (VZV). Foscarnet sodium exertsits antiviral activity by a selective inhibition at the pyrophosphntebinding site on virus-specific DNA polymerases and reversetranscriptases at concentrations that do not affect cellular DNApolymerases.

(c) Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide,trade name--VIRAZOLE™) is an antiviral drug provided as a sterile,lyophilized powder to be reconstituted for aerosol administration.Ribavirin is a synthetic nucleoside which is a stable, white,crystalline compound with a maximum solubility in water of 142 mg/ml at25° C. and with only a slight solubility in ethanol. The empiricalformula is C₈ H₁₂ N₄ O₅ and the molecular weight is 244.2 Daltons.Ribavirin has antiviral inhibitory activity in vitro against respiratorysyncytial virus, influenza virus, and herpes simplex virus. Ribavirin isalso active against respiratory syncytial virus (RSV) in experimentallyinfected cotton rats. In cell cultures, the inhibitory activity ofribavirin for RSV is selective. The mechanism of action is unknown.Reversal of the in vitro antiviral activity by guanosine or xanthosinesuggests ribavirin may act as an analogue of these cellular metabolites.

(d) Vidarabine (adenine arabinoside, Ara-A,9-β-D-arabinofuranosyladenine monohydrate, trade name--VIRA-A™) is anantiviral drug. Vidarabine is a purine nucleoside obtained fromfermentation cultures of Streptomyces antibioticus. Vidarabine is awhite, crystalline solid with the empirical formula, C₁₀ H₁₃ N₅ O₄.H₂ O.The molecular weight of vidarabine is 285.2, the solubility is 0.45mg/ml at 25° C., and the melting point ranges from 260° to 270° C.Vidarabine possesses in vitro and in vivo antiviral activity againstHerpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and in vitroactivity against varicella-zoster virus (VZV). The antiviral mechanismof action has not yet been established. Vidarabine is converted intonucleotides which inhibit viral DNA polymerase.

(e) Ganeiclovir sodium (9-(1,3-dihydroxy-2-propoxymethyl)guanine,monosodium salt, trade name--CYTOVENE™) is an antiviral drug activeagainst cytomegalovirus for intravenous administration. Ganeiclovirsodium has a molecular formula of C₉ H₁₂ N₆ NaO₄ and a molecular weightof 277.21. Ganeiclovir sodium is a white lyophilized powder with anaqueous solubility of greater than 50 mg/mL at 25° C. Ganeiclovir is asynthetic nucleoside analogue of 2'-deoxyguanosine that inhibitsreplication of herpes viruses both in vitro and in vivo. Sensitive humanviruses include cytomegalovirus (CMV), herpes simplex virus-1 and -2(HSV-1, HSV-2), Epstein-Barr virus (EBV), and varicella zoster virus(VZV).

(f) Zidovudine azidothymidine (AZT), 3'-azido-3'-deoxythymidine, tradename--RETROVIR™! is an antiretroviral drug active against humanimmunodeficiency virus (HIV) for oral administration. Zidovudine is awhite to beige, odorless, crystalline solid with a molecular weight of267.24 daltons and a molecular formula of C₁₀ H₁₃ N₅ O₄. Zidovudine isan inhibitor of the in vitro replication of some retroviruses includingHIV (also known as HTLV III, LAV, or ARV). Zidovudine is a thymidineanalogue in which the 3'hydroxy (--OH) group is replaced by an azido(--N3) group.

(g) Phenol (carbolic acid) is a topical antiviral, anesthetic,antiseptic, and antipruritic drug. Phenol is a colorless or whitecrystalline mass which is soluble in water, has a characteristic odor, amolecular formula of C₆ H₆ O, and a molecular weight of 94.11.

(h) Amantadine hydrochloride (1-adamantanamine hydrochloride, tradename--SYMMETREL™) has pharmacological actions as both an anti-Parkinsonand an antiviral drug. Amantadine hydrochloride is a stable white ornearly, white crystalline powder, freely soluble in water and soluble inalcohol and in chloroform. The antiviral activity of amantadinehydrochloride against influenza A is not completely understood but themode of action appears to be the prevention of the release of infectiousviral nucleic acid into the host cell.

(i) Interferon alfa-n3 (human leukocyte derived, trade name--ALFERON™)is a sterile aqueous formulation of purified, natural, human interferonalpha proteins for use by injection. Interferon alfa-n3 injectionconsists of interferon alpha proteins comprising approximately 166 aminoacids ranging in molecular weights from 16,000 to 27,000 daltons.Interferons are naturally occurring proteins with both antiviral andantiproliferative properties.

Preferred antiviral agents to be employed may be selected from the groupconsisting of acyclovir, foscarnet sodium, ribavirin, vidarabine,ganeiclovir sodium, zidovudine, phenol, amantadine hydrochloride, andinterferon alfa-n3. In a preferred embodiment, the antiviral agent isselected from the group consisting of acyclovir, foscarnet sodium,ribavirin, vidarabine, and ganeiclovir sodium. In a more preferredembodiment, the antiviral agent is acyclovir.

The antiviral agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the antiviral agent and/or a further time-releaseform of the antiviral agent. Without being limited thereto, suchphysical forms include free forms and encapsulated forms, and mixturesthereof.

The amount of antiviral agent used in the present invention may varydepending upon the therapeutic dosage recommended or permitted for theparticular antiviral agent. In general, the amount of antiviral agentpresent is the ordinary dosage required to obtain the desired result.Such dosages are known to the skilled practitioner in the medical artsand are not a part of the present invention. In a preferred embodiment,the antiviral agent in the antiviral-wound healing composition ispresent in an amount from about 0.1% to about 20%, preferably from about1% to about 10%, and more preferably from about 2% to about 7%, byweight.

B. Methods For Making The Antiviral-Wound Healing Compositions OfEmbodiment Two (H.A-D+V)

The present invention extends to methods for making the therapeuticantiviral-wound healing compositions (II.A-D+V). In general, atherapeutic antiviral-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D) andan antiviral agent. In a first aspect of Embodiment Two (II.A+V), anantiviral-wound healing therapeutic composition is made by forming anadmixture of an antiviral agent and a wound healing compositioncomprising (a) a pyruvate, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids. In a second aspect of EmbodimentTwo (II.B+V), an antiviral-wound healing therapeutic composition is madeby forming an admixture of an antiviral agent and a wound healingcomposition comprising (a) a pyruvate, (b) a lactate, and (c) a mixtureof saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (II.C+V), an antiviral-wound healing therapeuticcomposition is made by forming an admixture of an antiviral agent and awound healing composition comprising (a) an antioxidant, and (b) amixture of saturated and unsaturated fatty acids. In a fourth aspect ofEmbodiment Two (II.D+V), an antiviral-wound healing therapeuticcomposition is made by forming an admixture of an antiviral agent and awound healing composition comprising (a) a lactate, (b) an antioxidant,and (c) a mixture of saturated and unsaturated fatty acids.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic antiviral-wound healing composition (II.A+V)which comprises the steps of admixing the following ingredients:

(A) a therapeutically effective amount of an antiviral agent; and

(B) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

C. Methods For Employing The Antiviral-Wound Healing Compositions OfEmbodiment Two (II.A-D+V)

The present invention extends to methods for employing the therapeuticantiviral-wound healing compositions (II.A-D+V). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with anantiviral-wound healing composition (II.A+V) which comprises the stepsof:

(A) providing a therapeutic antiviral-wound healing composition whichcomprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) contacting the antiviral-wound healing composition with the infectedwound.

D. The Augmented Antiviral-Wound Healing Compositions Of Embodiment Two(II.A-D+V+M)

In another aspect of Embodiment Two, the therapeutic antiviral-woundhealing compositions (II.A-D+V) of the present invention may be combinedwith medicaments useful for treating wounds (M) to form augmentedantiviral-wound healing compositions (II.A-D+V+M). In this embodiment,the combination of the antiviral-wound healing composition of thepresent invention and the medicament useful for treating wounds providesan augmented antiviral-wound healing composition having an enhancedability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents, other antiviralagents, antikeratolytic agents, anti-inflammatory agents, antifungalagents, respiratory bursting inhibitors (lactic acid, adenosine),inhibitors of prostaglandin synthesis (ibuprofen, aspirin, indomethacin,meclofenomic acid, retinoic acid, padimate O, meclomen, oxybenzone),steroidal anti-inflammatory agents (corticosteroids including syntheticanalogs), antimicrobial agents (neosporin ointment, silvadine),antiseptic agents, anesthetic agents (pramoxine hydrochloride,lidocaine, benzocaine), cell nutrient media, bum relief medications, sunbum medications, sunscreen agents, acne preparations, tretinoin, insectbite and sting medications, wound cleansers, wound dressings, scarreducing agents (vitamin E), dermatological agents, antihistamineagents, antibacterial agents, bioadhesive agents, and mixtures thereof,to further enhance the proliferation and resuscitation rate of mammaliancells. Preferably, the medicaments useful for treating wounds areselected from the group consisting of cytotoxic agents, antiviralagents, antikeratolytic agents, anti-inflammatory agents, antifungalagents, tretinoin, sunscreen agents, a buffering agent to maintain thepH of dermatitis in a range from about 5 to about 8 together with ananti-inflammatory agent, topical antihistamine agents, antibacterialagents, respiratory bursting inhibitors, inhibitors of prostaglandinsynthesis, antimicrobial agents, cell nutrient media, scar reducingagents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedantiviral-wound healing composition (II.A+V+M) which comprises:

(A) a therapeutic antiviral-wound healing composition which comprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extends to methods for making the augmentedantiviral-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic antiviral-woundhealing composition with the medicament useful for treating wounds toprepare the augmented antiviral-wound healing composition.

The present invention also extends to methods for employing theaugmented antiviral-wound healing compositions. In general, an augmentedantiviral-wound healing composition is employed by contacting thecomposition with a wound. In a preferred embodiment, the invention isdirected to a method for healing an infected wound in a mammal with anaugmented antiviral-wound healing composition (II.A+V+M) which comprisesthe steps of:

(A) providing a therapeutic augmented antiviral-wound healingcomposition which comprises:

(1) a therapeutically effective amount of an antiviral agent;

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(3) providing a medicament useful for treating wounds; and

(B) contacting the augmented antiviral-wound healing composition withthe infected wound.

The types of wounds which may be healed using the antiviral-woundhealing compositions and the augmented antiviral-wound healingcompositions of the present invention are those which result from aninfected injury which causes epidermal damage. The topical therapeuticcompositions may be used orally in the form of a mouth wash or spray toprotect and accelerate the healing of injured oral tissue.

Methods for healing a wound comprise topically administering thecompositions of the present invention directly to a wound site toincrease the healing rate of the wound. The composition is maintained incontact with the wound for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

E. Formulations Of The Antiviral-Wound Healing Compositions OfEmbodiment Two (II.A-D+V)

Once prepared, the inventive therapeutic antiviral-wound healingcompositions and augmented antiviral-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles (oral and non-oral) to prepare a wide variety ofpharmaceutical compositions. The pharmaceutically acceptable carrierswhich may be employed and the methods used to prepare the pharmaceuticalcompositions have been described above in connection with theformulations of the wound healing compositions of Embodiment One(I.A-D).

In a preferred embodiment, the invention is directed to anantiviral-wound healing pharmaceutical composition which comprises:

(A) a therapeutic antiviral-wound healing composition (II.A+V) whichcomprises:

(1) a therapeutically effective amount of an antiviral agent; and

(2) a wound healing composition which comprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of an antiviral-woundhealing composition (II.A+V) which comprises:

(1) an antiviral agent; and

(2) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier; and

(C) admixing the antiviral-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

F. Examples Of The Antiviral-Wound Healing Compositions Of EmbodimentTwo (II.A-D+V)

Study 1

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic antiviral-wound healing compositions of the presentinvention versus conventional wound healing compositions. The results ofthis study are illustrated in examples 1-21.

Two animal models were used to examine the ability of the wound healingcomponents to reduce lesion development, duration, and severity.Mathematical modeling was used to determine the ratio and concentrationsof wound healing components used in the animal models. In the guinea pigmodel, formulas #11 and #17 reduced lesion development, duration, andseverity scores significantly compared to the vehicle control, BLISTEX™,and Acyclovir. Acyclovir was the only compound that reduced viral titerssignificantly. In the mouse model, formulas #1, #15, and #16 reducedclinical symptoms compared to the vehicle and BLISTEX™. Acyclovir, inthe mouse model, reduced lesion development, duration and severity,producing the best results. Statistical analysis of the data haveconfirmed the results. In both models, the best formulas contain anequal ratio of Vitamin E and pyruvate and the higher levels of fattyacids. In the guinea pig model, formulas #11 and #17 contained 0.5% ofboth Vitamin E and pyruvate. In the mouse model, formulas #1 and #16contained 4.75% of the same actives. Deviation from these ratios reducedthe herpes lesion healing efficacy for both models.

Guinea Pig Studies

The purpose of these studies was to evaluate the activity of variousantioxidant preparations administered topically in a primary genitalHSV-2 infection of guinea pigs. Eighteen different preparationscontaining varying concentrations of three agents (vitamin E, pyruvicacid and fatty acids) were evaluated. Treatment was initiated at 48hours post-infection, which is 1-2 days before external genital lesionsbegin to appear. The commercial preparations of 5% acyclovir(ACV)polyethylene glycol (PEG) and medicated BLISTEX™ for cold soreswere utilized as internal controls.

Materials and Methods

I. Medications

The experiment was placebo-controlled and the preparations were testedin a coded fashion (except for the ACV and BLISTEXTM).

II. Genital HSV-2 Infection of Guinea Pigs

A. Description of Model

Intravaginal inoculation of weanling guinea pigs with HSV-2 results in aprimary genital infection characterized by initial replication of virusin the vaginal tract followed by the development of external vesicularlesions. Virus titers peak on days one to three in the vaginal tract andgradually clears by days 7-10. The external genital lesions first appearon day four, peak lesion severity occurs on days 6-8, and the lesionsgenerally heal by days 15-18.

B. Virus and Viral Inoculation

The MS strain of HSV-2 was utilized for animal inoculation. FemaleHartley guinea pigs (Charles River, Kingston, N.Y.) weighing 250-300 gwere inoculated intravaginally (i.vag.) with approximately 1.2×10⁵plaque forming units one hour after being swabbed for removal of vaginalsecretions. Viral inoculation was accomplished by inserting a swabsoaked with virus into the vaginal tract and rotating about six times.

C. Treatment of Guinea Pigs

Groups of 6 guinea pigs were run in duplicate, consecutive experiments(with the exception of group 21, which was run only once). The guineapigs were treated on the external genital skin with 0.1 ml of eachpreparation, four times daily for ten days beginning 48 hours post-viralinoculation.

D. Sample Collection and Virus Assays

To determine the effect of treatment on HSV-2 replication in lesions,swabs of lesions were obtained during the primary infection on days 3,4, 5, 6, 7, and 10 after HSV-2 inoculation. The swabs were placed intubes containing 2.0 ml of media, and frozen at -70° C. until titratedfor HSV. To identify the number of animals that became infected, vaginalswabs were obtained from all animals on day and handled as above. Whenall samples were collected, they were thawed, vortexed, dilutedserially, and HSV-2 titers determined in rabbit kidney cells using amicrotiter CPE assay.

E. Scoring of External Lesions

To determine the effect of therapy on the development, spread, andhealing of external genital lesions, lesion severity was scored on a0-5 + scale through the primary infection (Table 7 and FIGS. 14-18).

F. Evaluation of Efficacy

The data for each of the two experiments were first analyzed separately,then the results were combined and reanalyzed.

Peak lesion scores, peak lesion virus titers, areas under lesionscore-day, and areas under the virus titer-day curves betweenplacebo-treated and drug-treated animals were compared using theMann-Whitney U rank sum test. A p-value of 0.05 or less was consideredsignificant.

Hairless Mouse Studies p This model was used to assess the ability ofvarious anti-oxidant compounds, applied topically to the infected area,to modify the clinical course of the infection.

Materials and Methods

Mice: six to eight week old male SKH-1 hairless mice (Charles River)were infected with HSV-1 virus, McIntyre strain. Infection was achievedunder general anesthesia (Ketamine, Xylazine) by the abrasion of a 1 cmsquare area (using a 25 gauge needle) centrally located on the dorsalsurface of a mouse. Virus was then applied directly onto the abradedarea (10 μl of a 1×10⁹ PFU/ml virus stock). Following inoculation of10-10⁶ PFU of HSV-1 McIntyre strain by scarification of the epidermis,herpetic lesions developed by day 5 and persisted through day 12 postinfection (p.i.). The viral lesions spread in a zosteriform pattern fromthe site of inoculation (midline on the back) to the abdominal area. Byday 10, lesions were crusted over and complete healing generallyoccurred by day 12 p.i. (post infection).

Individual mice were treated with test compounds starting on theafternoon of the infection day, and treatment was continued for 14 days.Treatments were administered at 7 a.m. and 4 p.m. each day and involvedthe use of a sterile cotton tipped applicator in such a manner that theaffected area was evenly coated with the test compound. If no lesionswere visible, only the site of infection was treated. Data, includinglesion scores, number of lesions, and lesion areas were recorded duringthe 7 a.m. treatment session. Each animal was recorded as having one offive possible scores: 0, no signs; 1, redness and swelling; 2, singlelesion; 3, multiple lesions; 4, a spread of lesions in a dermatomepattern (see FIGS. 19A-19D). In addition, the actual lesion area on theskin was measured using a micrometer (x and y axial values for eachlesion were obtained in millimeters and then multiplied together to givethe lesion area). For analysis, the individual lesions scores or areaswithin a treatment group were averaged on a daily basis.

Nineteen compounds with varying amounts of anti-oxidant compounds weretested. Control compounds included Zovirax ointment, BLISTEXTM, andpolyethylene glycol m.w. 400. Each experimental compound was tested on atotal of eight to sixteen mice.

Statistical Evaluation Product Design/Product Group Allocation of theGuinea Pig and Mouse Models

The purpose of this study was to convey the product groups and theallocations of the animals necessary to evaluate the optimal combinationof the wound healing components (vitamin E, unsaturated fatty acids, andsodium pyruvate) in the presence of phenol and lidocaine both at 0.5% byweight. The range of the three components in the wound healing, all from0.5% to 9.0% by weight, were incorporated in the experimental design.The design is a two-cubed factorial with six star points and one centerpoint with the "control groups" BLISTEX™, Acyclovir and untreated. Theproduct groups are listed in a random order in the attachments (Table8). This order was random and was not changed.

The experimental variance is important for estimating the number ofanimals per product group (sample size) so that the resulting study willhave sufficient power to detect a clinically meaningful effect. Anestimate of this variance for the mouse study was obtained from therange of the clinical symptom scale. The use of eight (8) mice perproduct group should be sufficient to achieve 80% power to detect aneffect of 0.5 units when conducting a two-sided t test at the 0.05 levelof significance. The sample size for the mouse study was approximatedusing only the clinical symptoms scale. The power for detecting aclinically meaningful effect of the lesion size (total vesicular area)was not known.

The allocation of product in mice (Table 8) contains eight (8) mice perproduct group, except for the second use of Product Group Number 14which contains four (4) mice. There are two uses of Product GroupNumber 1. The numbers of the products groups in this table are the sameas those identified in Table 1 and were not changed, since the order ofthese product groups have been randomized. The side receiving the listedproduct group was also randomized. The person scoring the clinicalsymptoms and measuring the lesion size did not know which product groupwas used on which animal (blinding).

An estimate of the experimental variance for the guinea pig study wasobtained from the range of the lesion severity scale. The use of twelve(12) guinea pigs per product group should be sufficient to achieve 80%power to detect an effect of 0.5 units when conducting a two-sided ttest at the 0.05 level of significance. The sample size for the guineapig study was approximated using only the lesion severity scale. Thepowers for detecting clinically meaningful effects in the viral titer,time to healing, and other measures over time (days) were unknown.

The allocation of the guinea pigs to the product groups is given inTable 9 in two blocks of six (6) guinea pigs per product group. Noticethat there are two uses of Product Group Numbers 1 and 14. The numbersof the product groups in this table are the same as those identified inTable 1 and were not changed, since the order of these product groupshave been randomized. The person making the observations did not knowwhich product group was used on which animal (blinding).

If the actual experimental variance is less than the estimate used, thenthe study will be more powerful than stated. Alternatively, if theactual experimental variance is more than the estimate used, then thestudy will be less powerful than stated. The design and sample sizecalculations in this memorandum have been guided by information providedby the investigator.

Guinea Pig Model Results and Discussion

I. Effect of Topical Antioxidants on Lesion Viral Replication in GuineaPigs: First Study

The effect of topical antioxidants on lesion virus titers are shown inTable 1. There were no significant differences observed in lesion virustiter-day areas under the curve (AUC) between drug and placebo-treatedanimals.

II. Effect of Topical Antioxidants on Lesion Development in Guinea Pigs:First Study

The effect of topical antioxidants on lesion development are summarizedin Table 2. Only group 9 exhibited a significant reduction in the lesionscore-day AUC when compared to the vehicle treated animals (group 8).Groups 14a, 1a, 7, 18, 8, 10, 1b, 5, 4 (BLISTEX™), 2, 19, 15, 16 and 20had significantly greater lesion score-day AUC's when compared to theappropriate control group (group 13 or 8).

III. Effect of Topical Antioxidants on Lesion Viral Replication inGuinea Pigs: Second Study

The effect of topical antioxidants on lesion virus titers are shown inTable 3. Significant differences were observed in lesion virus titer-dayAUC's in groups 6 (ACV) and 20. It should be noted that group 20 hadonly 4 out of 6 animals with positive vaginal virus titers. Moderatereductions in lesion AUC's were also observed with groups 12 and 2(p-values of 0.06 and 0.0-7, respectively).

IV. Effect of Topical Antioxidants on Lesion Development In Guinea Pigs:Second Study

The effect of topical antioxidants on lesion development are summarizedin Table 4. Groups 11, 17, and 20 exhibited significant reductions inthe lesion score-day AUC when compared to the vehicle treated animals(group 8). Groups 14a, 1a, 18, 9, 5, 4 (BLISTEX™), 2, 15, 16, 3 and 21had significantly greater lesion score-day AUC's when compared to thevehicle treated animals (group 8). Groups 10 and 1b had moderatelygreater lesion AUC's (p-values of 0.07 and 0.06, respectively).

V. Effect of Topical Antioxidants on Lesion Replication in Guinea Pigs:Combined Results

The effect of topical antioxidants on lesion virus titers from thecombined results of the first and second studies are shown in Table 5.The only significant difference observed in lesion virus titer-day AUC'swas with group 6 (ACV). A moderate reduction in the lesion titer AUC wasshown with group 21 (p-value of 0.07).

VI. Effect of Topical Antioxidants on Lesion Development In Guinea Pigs:Combined Resales

The effect of topical antioxidants on lesion development from thecombined results of the first and second studies are summarized in Table6. Only groups 11 and 17 exhibited a significant reduction in the lesionscore-day AUC when compared to the vehicle treated animals (group 8).Groups 14a, 1a, 18, 8, 9, 10, 1b, 5, 4 (BLISTEX™), 2, 19, 15, 16, 3 and21 had significantly greater lesion score-day AUC's when compared to theappropriate control animals (groups 13 and 8).

VII. Discussion of Guinea Pig Results

Because of the large number of samples to be tested (22) and the need tocompare directly all of the samples at one time, the study was conductedas two identical experiments with six animals per group. The genitalinfection of guinea pigs is a natural infection and like any biologicalsystem, there is variability from animal to animal in the naturalhistory of the disease and the rate of progression through the variousstages of the primary infection. Due to this variability, a minimumgroup size of 10 guinea pigs per group was established in order tominimize the variability within each group.

In the first second and combined studies, there was excellentcorrelation for effect of treatment on virus titers in lesions. In fact,only the 5% ACV preparation significantly reduced viral replication inexternal genital lesions.

The effect of treatment with the various compounds on the developmentand severity of lesions on the external genital skin was more variablebetween the two experiments, however, almost all of the preparationsresulted in more severe lesions than the untreated control, the vehiclecontrol, or the group treated with ACV. One of the reference compounds(BLISTEX™, group 4) was consistently worse than the vehicle control.Groups 11 and 17 were the only preparations that significantly reducedthe lesion score compared with the vehicle control. Groups 7, 12, 20 and14b were neutral preparations in that they did not decrease or increasethe severity of the genital lesions. In contrast, groups 18, 10, 1b, 5,4, 2, 15, 16, 3 and 21 clearly resulted in significantly more severedisease than those that were untreated, treated with vehicle alone, orwith ACV-PEG. Analysis of the various components in each of theformulations identifies those materials that contribute to healing orexacerbation of disease severity and the information obtained will beused to construct a more optimal formulation.

Mouse Model Results and Discussion

All groups contained at least 8 animals by the conclusion of the study,however, as many as 16 animals at minimum, were infected for eachtreatment. Mice that did not show clinical signs for at least twoconsecutive days following inoculation of the virus, were considered tobe uninfected and were excluded from the study (the Acyclovir controlwas an exception as this positive control was expected to prevent viralreplication and reduce clinical signs). The infection rate varied from60% to 100% over the course of experiments.

By taking daily measurements of the lesions, a disease curve wasconstructed which consisted of 3 phases of the infection: incubation,log, and resolution. The data, presented as the average lesion area (insq. mm) for the positive (Acyclovir) and negative (PEG) drug treatmentcontrols from a representative experiment, are shown in FIG. 11. Theincubation period for this infection spanned day 0 to day 5 p.i.Measurable herpetic lesions developed in the PEG-treated group between 5and 6 days p.i. The severity of the lesions continued to increase in thelog phase of the infection through day 7 p.i., and peak clinical signsoccurred on day 8 p.i. The resolution phase of the infection occurredfrom day 9 through day 12 p.i. Mice treated with acyclovir showedminimum clinical signs, only 2 out of a total of 18 HSV-infected micedeveloped clinical signs.

In addition to measuring the lesion area on a daily basis, symptomscores from 0 to 4 (see Materials and Methods section) were alsorecorded (FIG. 12). As seen in FIG. 12, this curve tended to have a morebroad pattern. This was due to the fact that infected mice had clinicalsigns of infection, such as erythema and swelling, prior to thedevelopment of the actual herpetic lesions.

The "area under the curves" for the lesions area and the clinicalsymptoms was also calculated. These data proved a useful way ofexpressing the dynamics of the infectious disease process. In FIG. 13,the "area under the curve" (y axis) for the clinical symptoms for eachgroup (numbers on the x axis) and the control groups (PEG, Base orBLISTEX™), represented by the dotted lines, was compared. Data pointsbelow the dotted lines had lower average clinical symptom scores thanthe controls. This analysis showed that compounds 1, 15 and 16 had themost reduced clinical symptoms compared to the control groups.

Summary of Mouse and Guinea Pig Studies

In summary, the guinea pig model produced statistically and clinicallysignificant results, while the mouse model provided clinically but notstatistically significant results. The guinea pig study showed that theconcentration of wound healing components in formulas #11 and #17reduced lesion development, duration, and severity. Acyclovir reducedviral titers, but its effect on viral lesion development, duration, andseverity was worse than groups 11 and 17. In the mouse model, groups 1,15, and 16 reduced clinical symptoms compared to the control group.Acyclovir reduced lesion development, duration, and severity, producingthe best results. The mouse model which is generally used as a screenfor antiviral compounds, was modified in an attempt to broaden itssensitivity to differentiate among the test formula. Unfortunately,lesion size variations increased within each test group, producingnon-statistically significant results for that parameter. In spite ofthese unforeseen problems, some meaningful data was obtained from themouse model experiment. In both models the best formulas contained anequal ratio of Vitamin E and pyruvate and the higher levels of fattyacids. In the guinea pig model, formulas #11 and #17 contained 0.5% ofboth Vitamin E and pyruvate. In the mouse model, formulas #1 and # 16contained 4.75% of the both actives therefore suggesting that equal buthigher concentrations of the actives were needed for skin penetration,healing and cell resuscitation. Variation from these ratios reduced thehealing efficacy of the formulas in both models. Both animal studiesconfirmed previous results attained on the efficacy of wound healingcompositions to accelerate wound healing.

FIG. 11 is a graph illustrating the lesion area curves for mice infectedwith herpes simplex virus and treated with acyclovir (ACV, positive) andpolyethylene glycol (PEG, negative) . The x-axis represents days postinfection and the y-axis represents the average lesion area (mm²).

FIG. 12 is a graph illustrating the symptom score curves for miceinfected with herpes simplex virus and treated with acyclovir (ACV,positive) and polyethylene glycol (PEG, negative) . The x-axisrepresents days post infection and the y-axis represents the symptomscore.

FIG. 13 is a graph illustrating the area under the symptom score curvesby group for mice infected with herpes simplex virus. The x-axisrepresents the groups and the y-axis represents the area under thesymptom score curve by day 12. The clinical symptoms for each group arerepresented as numbers on the x axis and the control groups(polyethylene glycol, base, or BLISTEX™) are represented by dottedlines.

FIGS. 14A-14B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 1.0 and 1.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 15A-15B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 2.0 and 2.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 16A-16B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 3.0 and 3.5. Thescorings range from 0 to 4, with 4 being the worst.

FIGS. 17A-17B are photographs illustrating the scoring of cold sorelesions in guinea pig. The scorings illustrated are 4.0 and 0.0(control). The scorings range from 0 to 4, with 4 being the worst.

FIGS. 18A-18B are photographs illustrating the scoring of cold sorelesions in guinea pig. Animals in FIG. 18A were treated with formulas 11and 17. Animals in FIG. 18B were treated with BLISTEX™. The scoringsrange from 0 to 4, with 4 being the worst.

FIGS. 19A-19D are photographs illustrating the scoring of cold sorelesions in hairless mice. The scorings illustrated are 1.0, 2.0, 3.0 and4.0, respectively. The scorings range from 0 to 4, with 4 being theworst.

                                      TABLE 1    __________________________________________________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    First Study                   Lesion Virus            #Vaginal Virus                   Tier-Day    Mean Peak            Positive/                   Area        Lesion    Treatment.sup.a            #Inoculated                   Under Curve                         p-Value.sup.b                               Virus Titer                                      p-Value.sup.b    __________________________________________________________________________    Group 13            6/6    16.5  --    3.9    --    (untreated)    Group 11            6/6    18.2  .sup. NS.sup.c                               3.7    NS    Group 14a            6/6    16.6  NS    3.7    NS    Group 1a            6/6    18.1  NS    3.7    NS    Group 7 6/6    15.9  NS    4.1    NS    Group 12            6/6    17.7  NS    3.9    NS    Group 18            6/6    18.7  NS    4.1    NS    Group 6 6/6    9.9   NS    4.0    NS    (5% ACV/PEG)    Group 8 6/6    19.5  NS    4.5    NS    Group 9 6/6    15.6  NS    3.8    NS    Group 10            6/6    17.0  NS    3.8    NS    Group 1b            6/6    18.4  NS    4.4    NS    Group 5 6/6    18.6  NS    4.1    0.08    Group 4 6/6    16.1  NS    4.0    0.07    (BLISTEX ™)    Group 2 6/6    20.0  NS    4.4    NS    Group 19            6/6    17.4  NS    3.9    0.07    Group 15            6/6    19.1  NS    4.3    NS    Group 16            6/6    19.2  NS    4.3    NS    Group 17            6/6    17.3  NS    4.2    NS    Group 3 6/6    20.0  NS    4.8    NS    Group 20            6/6    19.4  NS    4.4    NS    Group 14b            6/6    19.2  NS    4.4    NS    __________________________________________________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control anumals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).

                  TABLE 2    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    First Study              Lesion Score              Day Area              Area               Mean Peak    Treatment.sup.a              Under Curve                        P-Value.sup.b                                 Lesion Score                                          p-Value.sup.b    ______________________________________    Group 13  23.3      --       2.7      --    (untreated)    Group 11  26.2      .sup. NS.sup.c                                 2.8      NS    Group 14a 38.6      <0.05.sup.d                                 3.9      NS    Group 1a  38.5      <0.01    3.5      NS    Group 7   37.3      <0.01    3.2      NS    Group 12  28.4      NS       3.0      NS    Group 18  46.8      <0.001   3.9      NS    Group 6   31.6      NS       3.2      NS    (5% ACV/PEG)    Group 8   30.5      <0.01.sup.e                                 3.5      NS    Group 9   22.8      <0.05    2.8      NS    Group 10  40.0      <0.01    3.4      NS    Group 1b  45.1      <0.01    4.1      NS    Group 5   40.2      <0.01    3.5      NS    Group 4   37.4      <0.05    3.5      NS    (BLISTEX ™)    Group 2   40.8      <0.01    3.5      NS    Group 19  40.2      <0.01    3.3      <0.05    Group 15  46.4      <0.01    4.3      <0.05    Group 16  42.4      <0.001   3.7      NS    Group 17  25.2      NS       3.2      NS    Group 3   34.7      NS       3.4      NS    Group 20  47.9      <0.01    4.0      NS    Group 14b 25.7      NS       3.0      NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion severity was increased significantly compared to the     appropriate control animals (group 8 or group 13).     .sup.e Lesion severity was decreased significantly compared to vehicle     treated animals (group 8).

                                      TABLE 3    __________________________________________________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    Second Study                   Lesion Virus            #Vaginal Virus                   Titer-Day   Mean Peak            Positive/                   Area        Lesion    Treatment.sup.a            #Inoculated                   Under Curve                         P-Value.sup.b                               Virus Titer                                     P-Value.sup.b    __________________________________________________________________________    Group 13            6/6    9.8   --    2.7   --    (untreated)    Group 11            6/6    10.6  .sup. NS.sup.c                               2.7   NS    Group 14a            6/6    13.4  NS    3.6   NS    Group 1a            6/6    9.5   NS    3.0   NS    Group 7 6/6    10.3  NS    2.9   NS    Group 12            6/6    8.6   0.06.sup.d                               3.1   NS    Group 18            6/6    13.8  <0.001                               3.3   0.07    Group 6 6/6    2.3   NS    1.6   0.06.sup.e    (5% ACV/PEG)    Group 8 6/6    11.9  NS    3.6   NS    Group 9 6/6    9.8   NS    2.8   NS    Group 10            6/6    10.9  NS    2.8   NS    Group 1b            6/6    11.1  NS    3.0   NS    Group 5 6/6    12.8  NS    3.3   NS    Group 4 6/6    9.5   0.07  2.9   NS    (BLISTEX ™)    Group 2 6/6    8.9   NS    2.6   <0.05    Group 19            6/6    11.6  NS    3.2   NS    Group 15            6/6    13.1  NS    3.6   NS    Group 16            6/6    11.9  NS    2.7   0.07    Group 17            6/6    9.1   NS    2.7   0.07    Group 3 6/6    9.4   NS    2.3   <0.05    Group 20            6/6    5.7   <0.01 2.2   NS    Group 14b            6/6    12.7  NS    3.2   NS    Group 21            12/12  10.5  NS    3.0   NS    __________________________________________________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 mls applied topically to     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).     .sup.e Lesion titer was increased compared to untreated control animals     (group 13).

                  TABLE 4    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    Second Study              Lesion Score              Day Area              Area                Mean Peak                                          P-    Treatment.sup.a              Under Curve                        P-Value.sup.b                                  Lesion Score                                          Value.sup.b    ______________________________________    Group 13  21.7      --        2.4     --    (untreated)    Group 11  16.5      <0.65.sup.c                                  2.0     0.08    Group 14a 28.7      <0.05.sup.d                                  3.0     .sup. NS.sup.e    Group 1a  38.6      NS        3.8     <0.05    Group 7   24.5      NS        2.5     NS    Group 12  28.8      NS        2.7     NS    Group 18  35.0      <0.01     3.0     NS    Group 6   24.0      NS        2.2     NS    (5% ACV/PEG)    Group 8   25.0      NS        2.8     NS    Group 9   41.6      <0.001    3.5     NS    Group 10  29.1      <0.07     3.0     NS    Group 1b  28.5      <0.06     2.7     NS    Group 5   37.8      0.001     3.3     NS    Group 4   39.5      0.001     3.8     NS    (BLISTEX ™)    Group 2   37.9      <0.001    3.4     NS    Group 19  27.0      NS        2.4     NS    Group 15  50.7      <0.001    4.3     0.01    Group 16  32.7      <0.01     3.1     NS    Group 17  10.3      <0.001    1.7     <0.01    Group 3   36.5      0.001     3.3     NS    Group 20  11.6      <0.001    1.5     0.06    Group 14b 28.5      NS        2.8     NS    Group 21  35.6      <0.01     3.2     NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Lesion severity was decreased significantly compared to vehicle     animals (group 8).     .sup.d Lesion severity was increased significantly compared to vehicle     animals (group 8).     .sup.e Not significantly different from group 8 or group 13.

                                      TABLE 5    __________________________________________________________________________    Evaluation of Topical Antioxidants on Lesion Virus Titers    in a Primary Genital HSV-2 Infection in Guinea Pigs:    Combination of Results From the First and Second Studies                   Lesion Virus            #Vaginal Virus                   Titer-Day   Mean Peak            Positive/                   Area        Lesion    Treatment.sup.a            #Inoculated                   Under Curve                         p-Value.sup.b                               Virus Titer                                     p-Value.sup.b    __________________________________________________________________________    Group 13            12/12  13.1  --    3.3   --    (untreated)    Group 11            12/12  14.4  .sup. NS.sup.c                               3.5   NS    Group 14a            12/12  15.0  NS    3.7   NS    Group 1a            12/12  13.8  NS    3.3   0.07    Group 7 12/12  13.1  NS    3.5   NS    Group 12            12/12  13.1  NS    3.5   NS    Group 18            12/12  16.3  NS    3.7    Group 6 12/12  6.1   <0.01.sup.d                               2.8   NS    (5% ACV/PEG)    Group 8 12/12  15.7  NS    4.1   <0.05e    Group 9 12/12  12.7  NS    3.3   0.05.sup.e    Group 10            12/12  14.0  NS    3.3   NS    Group 1b            12/12  14.7  NS    3.7   NS    Group 5 12/12  15.7  NS    3.7   NS    Group 4 12/12  12.8  NS    3.5   0.07    (BLISTEX ™)    Group 2 12/12  14.5  NS    3.5   NS    Group 19            12/12  14.5  NS    3.5   NS    Group 15            12/12  16.1  NS    4.0   NS    Group 16            12/12  15.6  NS    3.5   NS    Group 17            12/12  13.2  NS    3.4   NS    Group 3 12/12  14.7  NS    3.6   NS    Group 20            12/12  12.6  NS    3.3   NS    Group 14b            12/12  15.8  NS    3.7   NS    Group 21            12/12  10.5  0.07  3.0   <0.01    __________________________________________________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion titer was decreased compared to vehicle treated animals     (group 8).     .sup.e Lesion titer was increased compared to untreated control animals     (group 13).

                  TABLE 6    ______________________________________    Evaluation of Topical Antioxidants on Lesion Severity    in a Primary Genital HSV-2 Infection of Guinea Pigs:    Combination of Results From the First and Second Studies              Lesion Score              Day Area              Area                Mean Peak                                          P-    Treatment.sup.a              Under Curve                        P-Value.sup.b                                  Lesion Score                                          Value.sup.b    ______________________________________    Group 13  22.5      --        2.5     --    (untreated)    Group 11  21.1      <0.05.sup.c                                  2.4     0.07    Group 14a 33.4      <0.05.sup.d                                  3.5     .sup. NS.sup.e    Group 1a  38.5      <0.01     3.5     NS    Group 7   30.5      NS        2.8     NS    Group 12  28.6      NS        2.6     NS    Group 18  40.9      <0.001    3.5     NS    Group 6   27.8      NS        2.7     NS    (5% ACV/PEG)    Group 8   27.4      <0.05     3.1     NS    Group 9   32.7      <0.05     3.2     NS    Group 10  34.9      <0.01     3.2     NS    Group 1b  37.4      <0.01     3.4     NS    Group 5   39.0      0.001     3.4     NS    Group 4   38.6      <0.01     3.7     NS    (BLISTEX ™)    Group 2   39.4      <0.001    3.5     NS    Group 19  32.9      <0.05     2.9     <0.001    Group 15  48.6      <0.001    4.3     NS    Group 16  36.7      <0.01     3.4     0.06    Group 17  17.6      <0.01     2.4     NS    Group 3   35.6      <0.01     3.4     NS    Group 20  28.8      NS        2.8     NS    Group 14b 27.3      NS        2.9     NS    Group 21  35.6      <0.01     3.2     NS    ______________________________________     .sup.a Treatment was initiated at 48 hours postinoculation. Animals were     treated 4 times per day for 10 days with 0.1 ml applied topically on     external genitalia.     .sup.b All groups were compared with group 8 (vehicle only), except for     group 8 which was compared with the untreated control animals (group 13).     .sup.c Not significantly different from group 8 or group 13.     .sup.d Lesion severity was increased significantly compared to the     appropriate control animals (group 8 or group 13).     .sup.e Lesion severity was decreased significantly compared to vehicle     treated animals (group 8).

                  TABLE 7    ______________________________________    Lesion Scoring System for a    Primary Genital HSV Infection in Guinea Pigs    Score Description    ______________________________________    0.0   Nothing, normal genital skin    0.5   Distinct erythema    1.0   1-2 lesions    1.5   3-5 lesions    2.0   More than 5 lesions    2.5   More than 5 lesions with some coalesced    3.0   Half of the area covered with coalesced lesions    3.5   Greater than half the area covered with coalesced lesions    4.0   As above with some ulceration (less than half the area ulcerated)    4.5   Half of the area ulcerated    5.0   Greater than half the area ulcerated    4.5   As above but with less than half the area crusted (scabbed over)    4.0   Greater than half of the area crusted/scabbed    3.5   As above but with loss of crust on less than half the area    3.0   Loss of crust on half to 3/4 of the area    2.5   Some distinct (larger patches) crust still left    2.0   Less than above but greater than 5 small areas of crust    1.5   3-5 small areas of crust left    1.0   1-2 small areas of crust left    0.5   Distinct erythema    0.0   Nothing, normal genital skin    ______________________________________

                  TABLE 8    ______________________________________    Cold Sore Product Groups in Mice and Guinea Pigs    Amount in Product (%)    Product        Unsaturated                             Sodium    Group Vitamin E                   Fatty Acids                             Pyruvate Phenol                                            Lidocaine    ______________________________________     1    4.75     4.75      4.75     0.5   0.5     2    9.00     9.00      9.00     0.5   0.5     3    0.50     0.50      9.00     0.5   0.5     4    BLISTEX ™     5    9.00     0.50      0.50     0.5   0.5     6    Acyclovir     7    4.7      0.00      4.75     0.5   0.5     8*   0.00     0.00      0.00     0.0   0.0     9    0.50     9.00      9.00     0.5   0.5    10    4.75     4.75      0.00     0.5   0.5    11    0.50     0.50      0.50     0.5   0.5    12    9.92     4.75      4 75     0.5   0.5    13    Untreated    14    0.00     0.00      0.00     0.5   0.5    15    4.75     4.75      9.92     0.5   0.5    16    4.75     9.92      4.75     0.5   0.5    17    0.50     9.00      0.50     0.5   0.5    18    9.00     9.00      0.50     0.5   0.5    19    9.00     0.50      9.00     0.5   0.5    20    0.00     4.75      4.75     0.5   0.5    ______________________________________     *base

                  TABLE 9    ______________________________________    Cold Sore Product Guinea Pig Allocation    Block      Product Group                          Guinea Pig Number    ______________________________________    1          13         1-6               11          7-12               14         13-18               1          19-24               7          25-30               12         31-36               18         37-42               6          43-48               8          49-54               9          55-60               10         61-66               1          67-72               5          73-78               4          79-84               2          85-90               19         91-96               15          97-102               16         103-108               17         109-114               3          115-120               20         121-126               14         127-132    2          13         133-138               11         139-144               14         145-150               1          151-156               7          157-162               12         163-168               18         169-174               6          175-180               8          181-186               9          187-192               10         193-198               1          199-204               5          205-210               4          211-216               2          217-222               19         223-228               15         229-234               16         235-240               17         241-246               3          247-252               20         253-258               14         259-264    ______________________________________

Statistical Analysis

The product (treatment) groups necessary to evaluate the optimalcombination of the components of wound healing (vitamin E, unsaturatedfatty acids, and sodium pyruvate) in the presence of phenol andlidocaine both at 0.5% by weight are incorporated in the experimentaldesign. The design is a two-cubed factorial with six star points and onecenter point with the "control groups" BLISTEX™, Acyclovir, anduntreated. The ranges of the three components in wound healing are allfrom 0.5% to 9.0% by weight. The product groups are listed in the randomorder presented to the investigator by number without any otheridentification.

The measures of efficacy considered for statistical analyses are thearea under the lesion score "curve", the peak lesion score, the time toresolution of the lesions, the area under the viral titer "curve", andthe peak viral titer. For each of these measures, the followingstatistical procedure was used. The treatment groups involving woundhealing were all compared to the treatment groups BLISTEX™, Acyclovir,and base (Treatment Groups 4, 6, and 14, respectively). The factorialportion of the treatment groups were then investigated to determine aresponse model for the components of wound healing. The model was thenused to predict what response could be expected for combinations of thewound healing components that were not statistically different from theminimum response.

Statistical Analysis of the Guinea Pig Model

The guinea pigs were allocated to the product groups in a random orderto two blocks of six (6) guinea pigs per product group with repeats ofProduct Group Numbers 1 and 14 (design center point and base,respectively). The lesion scores for. guinea pigs were recorded daily ondays 3 through 19 on a scale of 0 to 5 in half-unit increments inincreasing severity, while the titers were recorded on days 3, 4, 5, 6,7, and 10 as actually measured on a continuous scale. The areas underthe lesion and titer "curves" were calculated with the application ofthe trapezoidal rule. Any missing values were considered as zero valueson both scales. The peaks are the maximum values over the days observed.The time to resolution (healing time) of the lesions was defined asmidway between the last day of non-zero responses and zero responses. Ifno resolution occurred in the time frame of the study, the time toresolution was the last day plus a half day.

For area under the lesion score "curve", Treatment Groups 11 and 17 werestatistically different from BLISTEX™ (4) (p values of 0.0160 and0.0034, respectively), Treatment Group 17 was almost statisticallydifferent from base (14) (p value of 0.0502), and no treatment groupswere statistically different from Acyclovir (6). For peak lesion score,Treatment Groups 6, 7, 11, 12, 17, and 20 were statistically differentfrom BLISTEX™ (p values of 0.0178, 0.0479, 0.0031, 0.0479, 0.0031, and0.0297, respectively), Treatment Groups 11 and 17 were statisticallydifferent from base (p values of 0.0347 and 0.0347, respectively), andno treatment groups were statistically different from Acyclovir, exceptBLISTEX™. For time to lesion resolution, Treatment Groups 11, 17, and 20were statistically different from BLISTEX™ (p values of 0.0185, 0.0099,and 0.0283, respectively), Treatment Groups 11, 17, and 20 werestatistically different from base (p values of 0.0001, 0093, and 0.0312,respectively), and no treatment groups were statistically different fromAcyclovir. For area under the viral titer "curve", no treatment groupswere statistically different from BLISTEX™ and base, except Acyclovir,and all treatment groups were statistically different from Acyclovir.For peak viral titer, no treatment groups were statistically differentfrom BLISTEX™ and base, and Treatment Groups 1, 3, 5, 8, 12, 14, 15, 18,19, and 21 were statistically different from Acyclovir (p values of0.0195, 0.0342, 0.0128, 0.0005, 0.0479, 0.0035, 0.0009, 0.0098, 0.0383,and 0.0174, respectively).

Statistical Analysis of the Mouse Model

The mice were planned to be allocated to the treatment (product) groupsin a random order to one block of eight (8) mice per product group withrepeats of Product Group Numbers 1 and 14 (design center point and base,respectively). The lesion scores for mice were recorded daily on days 1through 14 on a scale of 0 to 4 in half-unit increments in increasingseverity and the lesion areas were measured on a continuous scale onthese same days. The areas under the lesion area and score "curves" werecalculated with the application of the trapezoidal rule. Any missingvalues were considered as zero on both scales. The peaks are the maximumvalues over the days observed. The time to resolution of the lesions wasdefined as midway between the last day of non-zero lesion scores andzero lesion scores. If no resolution occurred in the time frame of thestudy, the time to resolution was the last day plus a half day. Theactual allocation of the mice to the treatment groups was accomplishedon four (4) exposure dates. This resulted in an unbalanced design acrossexposure days. The means presented in this report are adjusted for thisunbalance, probably resulting in an increase in expected variation ofthe measures.

For area under the lesion area "curve", no treatment groups werestatistically different from BLISTEX™ and base, except Acyclovir, andall treatment groups except Treatment Groups 5 and 15 were statisticallydifferent from Acyclovir. For peak lesion area, no treatment groups werestatistically different from BLISTEX™ and base, except Acyclovir, andall treatment groups except Treatment Groups 17 and 19 werestatistically different from Acyclovir. For area under the lesion score"curve", no treatment groups were statistically different from BLISTEX™and base, except Acyclovir, and all treatment groups were statisticallydifferent from Acyclovir. For peak lesion score, no treatment groupswere statistically different from BLISTEX™ and base, except Acyclovir,and all treatment groups were statistically different from Acyclovir.For time to resolution of lesion scores, no treatment groups werestatistically different from BLISTEX™ and base, and all treatment groupswere statistically different from Acyclovir (6).

Conclusions

Various combinations of the components of wound healing in theneighborhood of 9% unsaturated fatty acids, 0.5% sodium pyruvate, and0.5% vitamin E statistically reduced the severity, intensity, andduration of coldsore lesions in the guinea pig model in comparison toBLISTEX™, Acyclovir, and base.

Only Acyclovir statistically reduces these measures of efficacy in themouse model.

Summary Analysis Of The Data From

Study 1

Six to eight week old male SKH-1 hairless mice (Charles River) wereinfected with HSV-1 virus, McIntyre strain. Infection was achieved undergeneral anesthesia (Ketamine, Xylazine) by the abrasion of a 1 cm squarearea (using a 25 gauge needle) centrally located on the dorsal surfaceof a mouse. Virus was then applied directly onto the abraded area (10 μlof a 1×10⁹ PFU/ml virus stock). Following inoculation of 10×10⁶ PFU ofHSV-1 McIntyre strain by scarification of the epidermis, herpeticlesions developed by day 5 and persisted through day 12 post infection(p.i.). The viral lesions spread in a zosteriform pattern from the siteof inoculation (middle of the back) to the abdominal area. By day 10,lesions were crusted over and complete healing generally occurred by day12 p.i.

Individual mice were treated with test compounds starting on theafternoon of the infection day, and treatment was continued for 14 days.Treatments were administered at 7 a.m. and 4 p.m. each day and involvedthe use of a sterile cotton tipped applicator in such a manner that theaffected area was evenly coated with the test compound. If no lesionswere visible, only the site of infection was treated. Data includinglesion scores, number of lesions, and lesion areas were recorded duringthe 7 a.m. treatment session. Each animal was recorded as having one offive possible scores: 0, no signs; 1, redness and swelling; 2, singlelesion; 3, multiple lesions; 4, a spread of lesions in a dermatonepattern. In addition, the actual lesion area on the skin was measuredusing a micrometer (x and y axial values for each lesion were obtainedin millimeters and then multiplied together to give the lesion area).For analysis, the individual lesion scores or areas within a treatmentgroup were averaged on a daily basis. The scores are set out in theTable below.

    __________________________________________________________________________    TABLE of Results (II.A-D + V)                     3                 2   Mean Lesion                           4      5    1            Lesion                     Peak Area                           Sum of Lesion                                  Mean Peak                                        6    Treatment    Size                     Severity                           Peaks 0-4                                  Averages                                        Days to    Groups       (mm.sup.2)                     (mm.sup.2)                           Scale  Severity                                        Healing    __________________________________________________________________________    1 - Control  130.8                     51.1  17.5   3.38  12    2 - Pyruvate &                 110.4                     40.9  18.5   3.14  12    Vitamin E    3 - Vitamin E &                 100.2                     36.9  16.8   3.43  11.8    Fatty acids    4 - Pyruvate &                 128.7                     48.3  18.1   3.24  12    Fatty Acids    5 - Pyruvate &                 87.8                     32.2  15.2   3.01  11.9    Vitamin E & Fatty Acids    (wound healing composition)    __________________________________________________________________________

Inspection of the results clearly shows that the three component woundhealing composition treatment achieved the best results in four of thefive measures (lesion size, lesion severity, sum of lesion peaks, andlesion peak severity).

The following proposed model was established to determine unexpectedresults from the wound healing composition.

    Change from control=Vitamin E+Fatty Acids+Sodium Pyruvate+Synergy

where Vitamin E represents the single component contribution of VitaminE, Fatty Acids is the single component effect of Fatty Acids, SodiumPyruvate is the single component effect of Sodium Pyruvate, and Synergyis the unexplained wound healing composition synergy effect of the threecomponents together.

Applying the data set out above in column 2 of the Table of Results(II.A-D+M) in this model provides the following results.

Lesion Size From (Column 2) in the Table of Results.

Vitamin E+Sodium Pyruvate=110.4-130.8mm²

Vitamin E+Fatty Acids=100.2-130.8mm²

Fatty Acids+Sodium Pyruvate=128.7-130.8mm²

Vitamin E+Fatty Acids+Sodium Pyruvate+Synergy=87.8-130.8mm²

This model now provides a linear equation system with four equations andfour unknowns, which can be solved by standard mathematical techniquesto give:

Vitamin E=-24.5

Sodium Pyruvate=+4

Fatty Acids=-6.1

Synergy=-16.4

Solving for the remaining columns of the Table of Results (II.A-D+M) inthis model provides the following results.

    __________________________________________________________________________    TABLE of Results of Contribution of Each Component (II.A-D + V)                     3                 2   Mean Lesion                           4      5    1            Lesion                     Peak Area                           Sum of Lesion                                  Mean Peak                                        6    Individual   Size                     Severity                           Peaks 0-4                                  Averages                                        Days to    Effects      (mm.sup.2)                     (mm.sup.2)                           Scale  Severity                                        Healing    __________________________________________________________________________    1 - Vitamin E                 -24.5                     -10.8 -0.15  0.0   -0.1    2 - Pyruvate +4.0                     +0.6  +1.15  -0.2  +0.1    3 - Fatty Acids                 -6.1                     -3.4  -0.55  0.0   -0.1    5 - Pyruvate &                 -16.4                     -5.3  -2.75  -0.2  0.0    Vitamin E & Fatty Acids    (wound healing composition)    __________________________________________________________________________

Comparing the predicted effect of the wound healing composition with theactual effect of wound healing composition gives the difference ineffect of wound healing somposition and the % synergy difference set outbelow.

    __________________________________________________________________________    Table of Results Showing Contribution of Wound Healing Composition    (II.A-D + V)                     3                 2   Mean Lesion                           4      5                 Lesion                     Peak Area                           Sum of Lesion                                  Mean Peak                                        6                 Size                     Severity                           Peaks 0-4                                  Averages                                        Days to    1            (mm.sup.2)                     (mm.sup.2)                           Scale  Severity                                        Healing    __________________________________________________________________________    Predicted Effect of                 -26.6                     -13.6  +0.45  -0.2  0    Wound Healing Composition    Actual Effect of                 -43.0                     -18.9  -2.3   -0.4 +0.1    Wound Healing Composition    Difference in Effect of                 -16.4                      -5.3  -2.75  -0.2 +0.1    Wound Healing Composition    % Synergy Difference                  61%                      39%  611%   100%   0    __________________________________________________________________________

As shown in the Table of Results Showing Contribution of Wound HealingComposition (II.A-D+V), the wound healing composition reduced four ofthe five lesion measurements greater than was predicted for the threecomponent effect. The wound healing composition reduced lesion sizes 61%more than was predicted for the three component effect. The woundhealing composition reduced mean lesion peak are (severity) 39% greaterthan was predicted for the three component effect. The wound healingcomposition reduced the sum of lesion peaks 611% greater than waspredicted for the three component effect. The wound healing compositionalso reduced mean peak averages 100% greater than was predicted for thethree component effect. The wound healing composition did not reduce thedays to healing in this model because mice require approximately fivedays to develop lesions, three days to respond (inflammatory phase),leaving only four days to heal. The effect of the wound healingcomposition to reduce is small in this model because the wound healingcomposition is not an antiviral agent and can only enhance healing afterthe inflammatory phase is over. In these examples, the antiviral agentwas phenol, which does not effectively reduce viral titers, and lesionsdo not heal until the viral infection is reduced. In summary, the woundhealing composition provided greater results than was predicted for thethree component. The wound healing composition was synergistic inreducing viral lesions and severity but did not reduce times to healingin this model.

Examples Of The Antiviral-Wound Healing Compositions Of Embodiment Two(II.A-D+V)

Study 2

This study is a summary of findings from an evaluation of a cold soretreatment product conducted among 20 patients who presented with theonset of symptoms of herpes labialus. The results from this study areillustrated in examples 22-41 below.

Twenty subjects were enrolled and completed the expert study. Eachsubject was administered a lip balm containing the wound healingcomposition, 2% sodium pyruvate, 2% vitamin E acetate, and 4% chickenfat, in a Lubriderm lotion base. Enrollment was based on the presence ofvesiculation and a positive viral assay for herpes. Treatment with thestudy product began immediately following the positive viral assay andcontinued for up to two (2) weeks depending upon when full resolution,i.e, elimination of scabbing, occurred.

Table 1 (not shown) is a summary of daily diary responses which showsthat pain, itching, and swelling predominated over the first three (3)days (Baseline, Day 2 and Day 3) after vesicle formation had occurredand thereafter diminished. Pain was no longer present in any subject byDay 7 and in only one (1) subject by Day 5. Itching extended slightlyfurther with four (4) subjects still reporting mild itching by Day 6,two (2) subjects by Days 7 and 8, one (1) subject by Day 9 and nosubjects by Day 10. The pattern for swelling closely paralleled that forpain with only one (1) subject reporting mild swelling on Days 6 and 7.

Results of clinical examinations are summarized in Table 2 (not shown).It is important to note in examining these tables that apparentday-to-day fluctuations in data are due to 1) the every-other-day visitschedule and 2) the fact that subjects were not on the same visitschedule. (Depending upon the day of the week on which subjects wereenrolled, their return visits would be adjusted to accommodate theweekends). Thus, data can be analyzed only for general trends.

Results show that vesiculation persisted in approximately two-thirds(2/3) of the patients to Day 3 and thereafter diminished significantly.There was no evidence of ulceration occurring with any subject at anyinterval. Scabbing was present in more than half of the subjects by Day3 and persisted at a relatively high level through Day 8 after which itdiminished significantly. Full resolution was noted for seven (7)subjects on Day 8 with an additional six (6) subjects on Day 9 (twosubjects), Day 10 (three subjects) and Day 11 (one subject). Theremaining seven (7) subjects were found to be fully resolved on Day 13(four subjects), Day 14 (two subjects) and Day 15 (one subject). Whenasked at their examination visits to assess pain, six (6) subjectsreported mild pain on Day 3 with no pain reported by any subject beyondthat interval. The severity of pruritis diminished significantly fromBaseline (5 moderate and 2 severe) to Day 3 (1 moderate and no severe)with a few subjects reporting mild pruritis between Days 4 and 8.

Table 3 (not shown) is a summary of the comparison of resolution time asdetermined by clinical examination and by the patient's recollection ofhistorical resolution time from prior incidence. No advantage in time tocold sore resolution is evident from examination of this data. Thenumber of days required for scabbing to occur was also clinicallyobserved and, in those instances in which the patient defined resolutionas scab formation, there still appeared to be no advantage to the studyproduct.

Subjective responses to questionnaires are summarized in Table 4 (notshown). Thirteen (13) of 20 subjects regarded the product as excellent,six (6) as good and one (1) poor. Eleven of the 13 subjects who ratedthe product as excellent claimed that it worked fast or faster thanother medications. Fourteen of the study participants regarded the coldsore as not as bad as normal, five (5) regarded it as about the same andone (1) as worse than normal. Regarding relief of pain, discomfortand/or soreness, 12 of 20 subjects regarded it as excellent and eight(8) as good.

In conclusion, under the conditions employed in this study, there wereno apparent increases in healing rate as the result of the use of thestudy product in the treatment of cold sores. However, the product wasperceived by approximately two-thirds (2/3) of the patients to beexcellent and to work fast or faster than other medications. Inaddition, the majority of subjects perceived the cold sore to be not asbad as normal and rated the product as excellent for the relief of pain,discomfort and/or soreness. Finally only one (1) adverse event occurred,namely, one (1) subject who developed an additional cold sore lesion.However, the development of the additional lesion is not regarded asproduct-related.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

We claim:
 1. A therapeutic antiviral-wound healing composition whichcomprises a therapeutically effective amount of an antiviral agent and awound healing composition, wherein the wound healing compositioncomprises:(a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty wherein said fatty acids are those fatty acidsrequired for the resuscitation of injured mammalian cells; whereincomponents a, b, and c are present in amounts sufficient tosynergistically enhance wound healing.
 2. The composition according toclaim 1, wherein the antiviral agent is selected from the groupconsisting of acyclovir, foscarnet sodium, ribavirin, vidarabine,ganeiclovir sodium, zidovudine, phenol, amantadine hydrochloride, andinterferon alfa-n3.
 3. The composition according to claim 2, wherein theantiviral agent is acyclovir.
 4. The composition according to claim 1,wherein the pyruvate is selected from the group consisting of pyruvicacid, lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesiumpyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, methylpyruvate, α-ketoglutaric acid, pharmaceutically acceptable salts ofpyruvic acid, prodrugs of pyruvic acid, and mixtures thereof.
 5. Thecomposition according to claim 4, wherein the pyruvate is sodiumpyruvate.
 6. The composition according to claim 1, wherein saidantioxidant is Vitamin E acetate.
 7. The composition according to claim1, wherein the antiviral agent is present in the therapeutic woundhealing composition in an amount from about 0.1% to about 20%, by weightof the therapeutic wound healing composition.
 8. The compositionaccording to claim 1, wherein pyruvate is present in the therapeuticwound healing composition in an amount from about 10% to about 50%, byweight of the therapeutic wound healing composition.
 9. The compositionaccording to claim 1, wherein the antioxidant is present in thetherapeutic wound healing composition in an amount from about 0.1% toabout 40%, by weight of the therapeutic wound healing composition.
 10. Amethod for treating an infected wound in a mammal with anantiviral-wound healing composition which comprises administering to amammal in need thereof:a therapeutic antiviral-wound healing compositionwhich comprises:(1) a therapeutically effective amount of an antiviralagent; and (2) a wound healing composition which comprises:(a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof; (b) anantioxidant; and, (c) a mixture of saturated and unsaturated fatty acidswherein said fatty acids are those fatty acids required for theresuscitation of injured mammalian cells; wherein components a, b, and care present in synergistic amounts sufficient to synergistically enhancewound healing.
 11. An augmented antiviral-wound healing compositionhaving an enhanced ability to prevent and reduce injury to mammaliancells which comprises:(A) a therapeutic antiviral-wound healingcomposition which comprises:(1) a therapeutically effective amount of anantiviral agent; and (2) a wound healing composition which comprises:a)pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;(b) an antioxidant; and, (c) a mixture of saturated and unsaturatedfatty acids wherein said fatty acids are those fatty acids required forthe repair of cellular membranes and resuscitation of mammalian cells:wherein components a, b, and c are present in amounts sufficient tosynergistically enhance wound healing; and, (B) a medicament useful fortreating wounds.
 12. The augmented antiviral-wound healing compositionaccording to claim 11, wherein the medicament useful for treating woundsis selected from the group consisting of immunostimulating agents, otherantiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, acne treating agents, sunscreen, agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, respiratory bursting inhibitors, inhibitors ofprostaglandin synthesis, antimicrobial agents, antiseptic agents,anesthetic agents, cell nutrient media, burn relief medications, sunburn medications, insect bite and sting medications, wound cleansers,wound dressings, scar reducing agents, and mixtures thereof.
 13. Amethod for treating an infected wound in a mammal with an augmentedantiviral-wound healing composition which comprises administering to amammal in need thereof:(A) a therapeutic augmented antiviral-woundhealing composition which comprises:(1) a therapeutically effectiveamount of an antiviral agent; (2) a wound healing composition whichcomprises:(a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof; (b) an antioxidant; and, (c) a mixture of saturated andunsaturated fatty acids wherein said fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofinjured mammalian cells; wherein components a, b, and c present inamount sufficient to synergistically enhance wound healing; and (3) amedicament useful for treating wounds.
 14. An antiviral-wound healingpharmaceutical composition which comprises:(A) a therapeuticantiviral-wound healing composition which comprises:(1) atherapeutically effective amount of an antiviral agent; and (2) a woundhealing composition which comprises:(a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof; (b) an antioxidant; and, (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells; wherein components a, b, and c arepresent in amounts sufficient to synergistically enhance wound healing;and, (B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.
 15. The composition according to claim 1, wherein theantioxidant is selected from the group consisting of all forms ofVitamin A; all forms of carotene; all forms of Vitamin C; all forms ofVitamin E; Vitamin E esters which readily undergo hydrolysis to VitaminE; prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E;pharmaceutically acceptable salts of Vitamin A. carotene, Vitamin C, andVitamin E; and mixtures thereof.
 16. The composition according to claim1 wherein the mixture of saturated and unsaturated fatty acids isselected from the group consisting of animal and vegetable fats andwaxes.
 17. The composition according to claim 16, wherein the mixture ofsaturated and unsaturated fatty acids is selected from the groupconsisting of human fat, chicken fat, cow fat, sheep fat, horse fat, pigfat, and whale fat.
 18. he composition according to claim 17, whereinthe mixture of saturated and unsaturated fatty acids comprises lauricacid, myristic acid, myristoleic acid, pentadecanoic acid, palmiticacid, palmitoleic acid, margaric acid, margaroleic acid, stearic acid,oleic acid, linoleic acid, linolenic acid, arachidic acid, and gadoleicacid.
 19. The composition according to claim 1, wherein the mixture ofsaturated and unsaturated fatty acids is present in the therapeuticwound healing composition in an amount from about 10% to about 50%, byweight of the therapeutic wound healing composition.